TY - JOUR
AU1 - Shayea, Abdulaziz M, F
AU2 - Mousa, Alyaa M, A
AU3 - Renno, Waleed, M
AU4 - Nadar, Mohammed, Shaban
AU5 - Qabazard,, Bedoor
AU6 - Yousif, Mariam H, M
AB - Abstract Long-term diabetic patients suffer immensely from diabetic neuropathy. This study was designed to investigate the effects of hydrogen sulfide (H2S) on peripheral neuropathy, activation of microglia, astrocytes, and the cascade secretion of proinflammatory cytokines in the streptozotocin (STZ)-induced peripheral diabetic neuropathy rat model. STZ-induced diabetic rats were treated with the water-soluble, slow-releasing H2S donor GYY4137 (50 mg/kg; i.p.) daily for 4 weeks. Antiallodynic/antihyperalgesic activities were evaluated using different tests and histopathological changes and the expression of proinflammatory cytokines in the spinal cord were examined. GYY4137 treatment produced neuroprotective effects in the spinal cord of diabetic animals and modulated their sensory deficits. The treatment decreased allodynia (p < 0.05) and mechanical hyperalgesia (p < 0.01) and restored thermal hyperalgesia (p < 0.001) compared with diabetic rats. The treatment decreased the microglial response and increased astrocyte counts in spinal cord gray and white matter compared with untreated diabetic rats. Proinflammatory cytokines were reduced in the treated group compared with diabetic rats. These results suggest that H2S has a potentially ameliorative effect on the neuropathic pain through the control of astrocyte activation and microglia-mediated inflammation, which may be considered as a possible treatment of peripheral nerve hypersensitivity in diabetic patients. Astrocyte, Diabetic neuropathy, GYY4137, Microglia, Neurobehavioral, Neuroprotection INTRODUCTION Diabetes mellitus has become a global burden. The complications of type 1 diabetes lead to the increase of healthcare burden; the costs are estimated to be >$7000 per person annually (1). Type 1 diabetes mellitus (T1DM) is a chronic disease characterized by autoimmune destruction of the insulin-producing pancreatic β-cell, which leads to progressive insulin deficiency and resultant hyperglycemia (2). While type 1 diabetes has a robust genetic component, the high incidence may also be attributed to environmental triggers. One of the many leading causes of disability in the western world is diabetic peripheral neuropathy (DPN), which is associated with considerable morbidity and cost to society (3). DPN, caused by microvascular complications of DM, is attributed to chronic hyperglycemia affecting patients with both type 1 and type 2 diabetes. DPN progresses more rapidly, and its manifestations are more severe in type 1 diabetes (4). Approximately two-thirds of diabetic patients suffer from clinical or subclinical neuropathy (4). Nevertheless, the pathogenesis of DPN remains incomplete despite is frequency and toll on society. Nerve damage generally affects feet and legs before arms and hands but neuropathy is also evident as structural damage to the nerves that supply solid organs (5). The symptoms of neuropathy are variable and range from mild to severe. They include increased vibration and thermal perception thresholds that can progress to sensory loss (6). Patients may also develop abnormal sensations, such as paresthesias, allodynia, hyperalgesia, and instinctive pain that synchronizes with the loss of normal sensory function (6). DPN can also cause muscle weakness and the loss of reflexes that often lead to changes in mobility, gait, and balance (7). Glial cells are found in both the PNS (Schwann cells, satellite glia, perineurial glia) as well as the CNS (astrocytes, oligodendrocytes, microglia, and perivascular glia) (8). Diabetes affects all glial cells of the spinal cord, and effects on oligodendrocytes, astrocytes, and microglia have the potential to drive spinal sensitization mechanisms that amplify sensory input. These effects offer potential sites for therapeutic intervention. Activated microglia and astrocytes respond to and release many signaling molecules that have protective and pathological functions; these may involve cytokines, chemokines, and neuronal transmission changes and the release of nitric oxide (9). An interruption in the balance between proinflammatory and anti-inflammatory agents is found upon the activation of microglial cells (10). Microglial activation induces changes in both morphology and function and recent studies propose that microglial cells may be required in pain initiation (11). Microglial activation is characterized morphologically by an increase in cell number and cell body hypertrophy with thickened and retracted processes. This is accompanied by elevated levels of microglial markers, such as CD11b and ionized calcium-binding adapter molecule-1 (Iba1) (12). Following activation, microglia migrate and become phagocytic eliminating damaged or dead cells and toxic matter (13). Chronic activation of microglia in a broad range of CNS disorders results in their secretion of proinflammatory molecules including IL-1α, IL-1β, TNF-α, IL-6, IL-12, and chemokines (fractalkine; macrophage inflammatory protein-1α, and β) (14). Microglial activation also results in activation of the autonomic sympathetic nervous system and the hypothalamic-pituitary axis (15, 16). The CNS may then become excessively sensitive to pain (central sensitization). In a model of painful diabetic neuropathy induced by streptozotocin (STZ), activated microglia in the dorsal horn of the spinal cord were increased in number, expression of microglial markers, and exhibited a hypertrophic morphology that could last for 6 months. This activation was particularly prominent in the fourth lumbar (L4) segment of the dorsal horn (12). Hydrogen sulfide (H2S) was the third endogenous gaseous transmitter discovered after nitric oxide and carbon monoxide. It exerts various physiological effects in the body and has been linked to several pathological conditions (17). H2S was shown to exert a multitude of beneficial effects on neuronal, cardiovascular, respiratory and reproductive systems because of its anti-inflammatory, antioxidant and cytoprotective functions (18). Moreover, H2S has been shown to protect against diabetic nephropathy (19), and the development of neuropathic pain, possibly via activation of microglia in the spinal cord in mice (12). Here, we investigated the effects of the H2S donor GYY4137 on peripheral neuropathy, activation of astrocytes, microglia and proinflammatory cytokines, IL-1β, IL-6, and TNF-α, in the STZ-induced DPN rat model. MATERIALS AND METHODS Animals Sprague Dawley male rats (2–3 months of age, ∼300–400 g; n = 52) were obtained from the Animal Resources Center at Kuwait University. They were housed in plastic cages that had access to pelleted chow and water ad libitum (3 males/cage). The bedding in all cages was hardwood chips that provided them with a high absorbance rate as well as a comfortable environment for breeding. All the rats were kept under a 12-hour light/dark cycle (light from 7 AM to 7 PM). The temperature was set at 25°C to provide them with a warm environment (20, 21). Rats were randomly assigned to the following groups: group 1 (control; n = 10), group 2 (control + GYY4137; n = 10), group 3 (diabetic; n = 16), and group 4 (diabetic + GYY4137; n = 16). Drug GYY4137 is a water-soluble, slow-releasing H2S donor with potential vasodilation and anti-inflammatory properties. It has also been shown to inhibit the lipopolysaccharide-induced release of various proinflammatory mediators (22). Disease Induction For the induction of diabetes, the animals fasted overnight for 12 hours. Diabetes was induced by a single dose of STZ (50 mg/kg, i.p.) (Cat. No. 18883-66-4; Sigma-Aldrich, Taufkirchen, Germany) freshly dissolved in ice-cold citrate buffer pH 4.5. After STZ injection, the animals were allowed free access to food and water. Diabetes was checked after 48 hours by estimating the blood sugar levels using a glucometer (Bayer Corporation, Elkhart, IN). The animals with a blood sugar level of >300 mg/kg were considered diabetic and included in the study (23). Groups 1 and 3 were injected with normal saline daily, whereas groups 2 and 4 were injected with GYY4173 daily (24). All experimental procedures were in line with the guidelines set and approved by the Kuwait University Health Science Center Animal Research ethics committee. Neurobehavioral Sensory Tests Von Frey Filaments The tactile allodynia response of the animals was measured by assessing the 50% mechanical threshold for responses to stimuli applied to the hind paws of experimental animals after the second and fourth week of the experiment. Von Frey filaments are a series of nylon monofilaments that are graded in stiffness and have different bending forces, depending on their diameter, used to assess the sensitivity of the skin to tactile stimulation (25). A single filament can be brushed along the skin to generate a dynamic stimulus or applied statically at a single location 3 times to produce a static stimulus. Allodynia thresholds were assessed by using a complete kit of these filaments (Cat. No. 37450-275; Ugo Basile SRL, Italy), spanning a range of forces. In addition to the paw withdrawal reflex, reactions, such as licking, escaping, and head orientation, were also utilized as supraspinal indicators. The evaluation of the mechanical sensitivity of the paw was started by placing rats inside a clear plastic cage with a wire mesh bottom, which allows full access to the plantar surface of the paw. After an initial period of acclimatization, monofilaments with graded bending strength are pressed perpendicularly against the glabrous skin of the plantar surface of the paw with sufficient force to form a U-shape. The duration of a stimulus application varies. Sharp paw withdrawal and also immediate flinching upon removal of the hair are considered as a positive response (26–28). Ascending von Frey testing procedure detects the frequency of responses to a series of stimuli. In the absence of a response, the next stronger hair is presented, while if a response is present, the next lighter hair is used. Hotplate Hyperalgesia Test Hotplate hyperalgesia apparatus (Hot Cold Plate; Model No. 35150; Ugo Basile SRL, Italy) was used to measure thermal hyperalgesia. The rat was placed on a preheated plate (at 50°C) within the hotplate apparatus, as described previously (20, 21, 29, 30). The time required to observe the endpoint was recorded manually, and the average of 3 readings was taken. Behaviors, such as licking and head turn, were also utilized in an earlier study as indicators for supraspinal processing (27). This assay permits the minimum handling of rats. Paw Pressure Test The Paw Pressure test or the Randall-Selitto test is a technique for the measurement of pain response in animals (30, 31). Pain is considered to be present if the rat starts to exhibit the flight or struggle response (31). The animals were tested blindly on the second and fourth week following treatments using Randall-Selitto paw-pressure test (Model No. 37215; Ugo Basile SRL, Italy), as described earlier (21, 25, 29). The equation of left hind paw is (Left hind paw – Right hind paw/left hind paw * 100) and for right hind paw (Right hind paw – Left hind paw/Right hind paw * 100). Statistical Analysis All behavioral data were analyzed by 2-way repeated-measures ANOVA followed by Bonferroni’s F- and Fisher least significant difference comparison post hoc test or Student t-test, as appropriate. All statistical analysis was performed using SPSS (IBM SPSS Statistics V 21.0; IBM Corp. Armonk, NY) and p values <0.05 were considered statistically significant. Perfusion and Tissue Processing The animals were killed after 28 days of the experiment. Briefly, animals were anesthetized with chloral hydrate (400 mg/kg) administered intraperitoneally (i.p.). They were then transferred to a ventilated bench for intracardiac perfusion by opening the thorax to expose the heart and introducing a cannula into the left ventricle. At the same time, a cut was made in the upper anterior part of the right atrium to allow the blood to drain. After that, normal saline was first introduced and allowed to circulate to rinse the blood from the body. Then, freshly prepared 4% formaldehyde in 0.1 phosphate buffer (pH = 7.4) was introduced for tissue fixation. The perfusion solutions were introduced under pressure by suspending the solution bottles ∼1.5 m above the animal. Following fixation, the relevant parts of the spinal cord were removed from the animal by dissection. To confirm the identity of specific segments, specific landmarks were used in conjunction with the dissection of dorsal roots. The spinal segments were determined from the fifth lumbar spinal dorsal roots. The L5 DRG is just caudal to the rostral extent of the hip bones (ilia), and the L4 DRG lies just rostral to this landmark. These roots were followed to identify the corresponding segment and adjacent segments. After removal, the spinal cords were postfixed overnight in 4% paraformaldehyde. Before sectioning, the spinal cord was divided into blocks. Blocks were mounted on the microtome and transverse sections cut at 5-µm thickness. Sections were collected into tubes containing 0.1 M phosphate buffer, ready for performing immunohistochemistry. NeuN, GFAP, and Iba-1 Quantitative Immunohistochemistry Immunostaining Rat tissues were processed for immunohistochemistry as previously described (20, 32). The lumbar L3–L6 segments of the spinal cord where the sciatic nerve originates were collected and cut into 3 1-mm-thick sections. Random 5-μm-thick sections (10 sections/spinal cord) were then cut, mounted on Poly-l-lysine coated glass slides, and kept overnight for drying. A total of 10 sections/animal were used for each Iba-1, GFAP, and NeuN quantitative immunostaining (20, 29, 32). The sections were dewaxed for 30 minutes, and the slides were placed on a hot plate (set at 60°C). Then the slides were dipped twice in xylene for 5 minutes each. Rehydration was carried out by dipping the slides in absolute alcohol, 90% alcohol, 70% alcohol twice for 3 minutes each, respectively. The tissue slides were briefly washed in the distilled water. Antigen retrieval was done using boiling citrate buffer at 40°C for 15 minutes (citrate buffer: 2.94 g of sodium citrate in 1 L of distilled water, adjust pH = 6). After settling to room temperature, the sections were washed once with 0.1 M phosphate-buffered saline (PBS) for 5 minutes. The tissues were fixed adequately in cold methanol for 5 minutes and then washed with 0.1 M PBS for 5 minutes. Tissue slides kept in the moisture chamber were circled in using a PAP pen. Peroxidase blocking was done after incubating the sections in 3% H2O2 (Fluka Chemika, Buchs, Switzerland) at room temperature for 10 minutes to quench the endogenous peroxidase activity and washing with 0.1 M PBS for 5 minutes each twice. Sections were incubated for 30 minutes with appropriate blocking serum solution (5% normal horse serum [Cat. No. H0146; Sigma-Aldrich, St. Louis, MO]/normal goat serum [Cat. No. S26-M, Sigma-Aldrich] and 0.01% Triton X-100 in PBS) before applying the primary antibody for 60 minutes. The primary antibody solution was the recombinant anti-Iba1 antibody [EPR16589 (ab178847); Cambridge Biomedical Campus, Cambridge, UK] for the detection of the microglial marker, Iba1, at 1:8000 dilutions. The primary antibody solution was a mouse monoclonal to the GFAP anti-GFAP antibody (GA-5: sc-58766, mouse monoclonal—Santa Cruz Biotechnology, Dallas, TX) for the detection of astrocyte marker, GFAP, at 1:200 dilutions. Also, the primary antibody solution was mouse monoclonal to anti-NeuN (anti-NeuN, Clone A60, Mouse Monoclonal Antibody, Cat# MAB377, Millipore. Billerica, MA) for the detection of the neuron marker NeuN at 1:200 dilution. Sections were incubated with primary antibody overnight and washed with 0.1 M PBS thrice for 5 minutes each. Linking was performed through a multi-link antibody (a biotinylated immunoglobulin) for 20 minutes at room temperature and washed thoroughly with 0.1 M PBS thrice for 5 minutes each. Labeled sections with horseradish peroxidase-conjugated streptavidin for 20 minutes at room temperature and then again carried out washing with 0.1 M PBS thrice for 5 minutes each. Application of 3-diaminobenzidine (DAB) (DAB Kit, SK-4100, Vector Labs, Burlingame, CA) chromogen solution is made by incubating sections with DAB for 10 minutes (DAB solution: 2 drops DAB to 1 mL of DAB buffer) and washing again. Sections are counterstained with hematoxylin (Cat No. H9627-100G; Sigma) for 10 minutes and rinsed in tap water. Finally, slides are dehydrated in 70% alcohol, 90% alcohol, absolute alcohol twice for 3 minutes each, and then xylene twice for 5 minutes each. Sections were mounted using DPX (Fluka Chemika) and visualized using an Olympus microscope (DP-72; Olympus, Tokyo, Japan). NeuN-, Iba-1-, and GFAP-Immunostained Neuronal Count NeuN-immunoreactive neuronal counts in the dorsal horn and ventral horn regions of the spinal cord were done using the NIS-Elements D image analysis system (Nikon, Tokyo, Japan), as previously described (20, 32). A total of 10 sections per spinal cord were selected and analyzed as previously described (20, 32, 33). The region of the spinal cord under analysis was focused at 40× magnification, and an image was transferred to a computer monitor with a high-resolution digital Olympus camera (DP-72; Olympus) attached to an Olympus microscope. The neurons in the entire dorsal and ventral horn regions were counted. Slides were coded to avoid observer bias. The mean number of neurons per section was calculated for statistical analysis. Iba-1- and GFAP-immunoreactive staining intensities were measured with Cell Sens Dimension software (Cell Sens V3.1.; Olympus). From each rat, 10 sections were randomly selected for intensity measurement. The 10 sampling results per animal were averaged, then the averages of animal data per group (n = 10 for the control and control + GYY4137 groups; n = 16 for diabetic and diabetic + GYY4137 groups) were statistically analyzed. The total intensity in the entire white and gray matter was measured blindly, and the mean Iba-1- and GFAP-immunoreactive intensity per section was calculated for statistical analysis (20, 32). Western Blot The whole of L4–L6 lumbar spinal cord segments were quickly isolated, immediately frozen in liquid nitrogen, and stored at –70°C. Frozen tissues were homogenized in radioimmunoprecipitation assay buffer (RIPA Lysis buffer system; Cat. No. sc-24948, Santa Cruz Biotechnology). Immediately before lysing cells, 10 μL PMSF solution, 10 μL sodium orthovanadate solution, and 10–20 μL protease inhibitor cocktail solution were combined per mL of 1× RIPA Lysis buffer to prepare complete RIPA (per gram of tissue requires 3 mL of complete RIPA). The total protein in each homogenate sample was determined by using Epoch Microplate Spectrophotometer (Monochromator based microplate reader, BioTek, Winooski, VT). Four percent to 20% Mini-PROTEAN TGX Precast Protein Gel (Bio-Rad Laboratories, Cat. No. 4561093; Hercules, CA) was used for running the Western blot. Precision Plus Protein Kaleidoscope Standards (Bio-Rad Laboratories, Cat No. 1610375) was used as the ladder. Fifty microgram of the sample was loaded into each well and run under these parameters: voltage (100 V), 50–75 minutes. The PVDF membrane (Bio-Rad Laboratories, Cat. No. 162-0177) was soaked in methanol (10 minutes), distilled water (10 minutes), and 1× transfer buffer (#NP00061, Invitrogen) (10 minutes). The gel was transferred to the membrane at 75 V (2 gels) for 75-minute blotting. The membrane was blocked in blocking solution (5% nonfat milk-Regilait in TBS-T [mixture of Tween 20 and Tris-buffered saline]) for 1 hour at room temperature o1n a shaker. Western blotting determined the expression levels of various proteins (IL-1β, IL-6, Iba1, and TNF-α) after resolving the lysate on SDS-PAGE (Bio-Rad, Hercules, CA). The primary antibodies include rabbit polyclonal to IL-1β-anti-IL-1β antibody (Abcam Cat. No. ab9722; Abcam, Cambridge, United Kingdom), anti-TNF-α antibody-rabbit polyclonal to TNF alpha (Abcam Cat. No. ab6671), goat polyclonal to Iba1 (Abcam Cat. No. ab5076), and anti-IL-6 antibody-rabbit polyclonal to IL-6 (Abcam Cat. No. ab6672) incubated overnight at 4°C (Supplementary Data Table S1). Anti-rabbit IgG (whole molecule) peroxidase conjugate antibody produced in goat (Sigma Cat. No. A6154) was the secondary antibody and incubated for 2 hours. Monoclonal anti-β-actin antibody produced in mouse (Sigma Cat. No. A5441) was used as a loading control. Secondary antibody against actin was anti-mouse IgG (whole molecule) peroxidase antibody produced in rabbit (Sigma Cat. No. A9044) and kept for 2 hours. Membranes were washed thoroughly 6 times with TBS-T, 5 minutes each. In a dark room, ECL prime (GE Healthcare Amersham ECL Prime Western Blotting Detection Reagent [Amersham Cat. No. RPN2232]; Amersham, Buckinghamshire, UK) was uniformly added to the membrane and kept for 5 minutes. The membrane was placed in a Kapak pouch and kept under cassette with the film (GE Healthcare Amersham Hyperfilm ECL; Cat No. GE28-9068-35; Amersham) on top of the membrane. Blots were quantified using GS-800 Calibrated Image Densitometer (Bio-Rad Laboratories) (25). Data Analysis Images of the spinal cords obtained from 3 rats per group were analyzed. A total of 10 images from each section were digitized and stored in uncompressed tagged image file format (TIFF) with 24-bit RGB class and 640 × 480-pixel resolution. The 10 sampling results per animal were averaged then the averages of 5 animal data per group were statistically analyzed. All data are presented as the mean ± SEM. All calculations and all statistical procedures were performed using SPSS (version 21.0). The group values were compared using a Mann-Whitney U test. A p value of <0.05 was considered significant. RESULTS Effect of Treatment of STZ-Diabetic Rats With GYY4137 on Body Weight, Spinal Cord Weight, and Blood Glucose Levels Data from Figure 1A demonstrate that there was no significant difference between the 4 groups at week 1. However, at week 4 there was a significant (p < 0.001) increase in weights of both control and control + GYY4137-treated rats compared with diabetic groups. On the other hand, the diabetic rats treated with GYY4137 maintained a significant (p < 0.05) increase in body weight compared with the diabetic group in the fourth week of the experiment. FIGURE 1. Open in new tabDownload slide (A) Graph comparing the bodyweight of the different experimental groups. Normal rats showed increased body weight from week 1 to week 4 (*p < 0.001) compared with STZ-induced diabetes and STZ-induced diabetes + GYY 4137 groups. Also, STZ-induced diabetes + GYY 4137 rats showed continuous weight loss (**p < 0.05) compared with the STZ-induced diabetes group. Values are mean ± SE, n = 6. (B) Graph comparing the blood glucose level in the experimental groups at week 1 and week 4 post-STZ-induced diabetes. Diabetic and diabetic treated groups showed significantly *p < 0.001 higher blood glucose levels compared with control groups at week 1 and week 4, indicating hyperglycemia. Values are mean ± SE, n = 6. (C) At week 4, diabetic rats exhibited a significant increase in spinal cord weight compared with the rest of the groups (*p value < 0.0001). Also, the control rats with GYY 4137 and the diabetic group treated with GYY 4137 had a significantly higher increase in the weight of the spinal cord compared with the control group (**p < 0.0001). Values are mean ± SE, n = 6. FIGURE 1. Open in new tabDownload slide (A) Graph comparing the bodyweight of the different experimental groups. Normal rats showed increased body weight from week 1 to week 4 (*p < 0.001) compared with STZ-induced diabetes and STZ-induced diabetes + GYY 4137 groups. Also, STZ-induced diabetes + GYY 4137 rats showed continuous weight loss (**p < 0.05) compared with the STZ-induced diabetes group. Values are mean ± SE, n = 6. (B) Graph comparing the blood glucose level in the experimental groups at week 1 and week 4 post-STZ-induced diabetes. Diabetic and diabetic treated groups showed significantly *p < 0.001 higher blood glucose levels compared with control groups at week 1 and week 4, indicating hyperglycemia. Values are mean ± SE, n = 6. (C) At week 4, diabetic rats exhibited a significant increase in spinal cord weight compared with the rest of the groups (*p value < 0.0001). Also, the control rats with GYY 4137 and the diabetic group treated with GYY 4137 had a significantly higher increase in the weight of the spinal cord compared with the control group (**p < 0.0001). Values are mean ± SE, n = 6. The glucose level in all experimental groups did not show any significant difference during week 1 (p < 0.001) and week 4 (p < 0.001) (Fig. 1B). However, at week 4, both the diabetic and diabetic + GYY4137-treated groups showed a much higher increase in the blood glucose levels, whereas both control groups had normal glucose levels. At week 4 (after killing the animals), diabetic rats exhibited a substantially significant (p < 0.0001) increase in spinal cord weight compared with the rest of the groups (Fig. 1C). Also, the control + GYY4137 and the diabetic group treated with GYY4137 had a significantly (p < 0.0001) higher increase in spinal cord weighs compared with the control group. Neurobehavioral Sensory Tests Hotplate Test Hotplate tests were carried out for all experimental animals at week 2 and week 4 (Fig. 2A). The diabetic animals showed a significantly (p < 0.02) increased level of hotplate reflex latency in the second and fourth weeks of the experiment. However, the GYY4137-treated diabetic rats did not show a significant difference compared with the control groups at week 2 and week 4. Moreover, the GYY4137-treated diabetic group showed a significantly (p < 0.001) decreased level of thermal nociception latency compared with diabetic rats at weeks 2–4. FIGURE 2. Open in new tabDownload slide (A) Bar graph showing the results of hot plate analysis between all the experimental groups at week 2 and week 4 interval. (*p < 0.02) diabetic versus control, control + GYY 4137, and diabetic + GYY 4137 groups. (**p < 0.001) diabetic + GYY4137 versus diabetic group. (B) Bar graph showing the results of paw pressure analysis of the left hind limb between all the experimental groups at week 2 and week 4 intervals. At week 2, (*p < 0.017) diabetic group versus control group; (**p < 0.001) diabetic + GYY4137 group versus control group. At week 4, (*** p < 0.039) control groups versus diabetic group; (# p < 0.001) diabetic + GYY4137 group versus diabetic group. (C) Bar graph showing the results of paw pressure analysis of the right hind limb between all experimental groups at week 2 and week 4 intervals. At week 2, (*p < 0.009) diabetic group versus control groups; (**p < 0.002) diabetic + GYY 4137 group versus control groups. At week 4, (***p < 0.045) control group versus diabetic group; (****p < 0.001) diabetic + GYY 4137 group versus diabetic group. (D) Bar graph showing the results of Von Frey analysis between all the groups at week 2 and week 4 intervals. At week 4, (*p < 0.001) diabetic group versus control group; (**p < 0.05) diabetic + GYY 4137 group versus diabetic group. (E) Geometry graph showing the results of Von Frey analysis. The figure shows von Frey Test values for 5 animals each at weeks 2 and 4, for control, control with GYY4137, diabetic and diabetic with GYY4137 groups. In the controls, the values at week 2 varied from G10 to G180, and at week 4 from G10 to G60. The threshold was around G60. In the controls + GYY4137 group, the values at week 2 varied from G60 to G180, while at week 4 all the values were constant at G60. Hence, the threshold was good at G60. In the diabetic group, the values at week 2 varied from G6 to G60, and at week 4, the values declined to a level between G4 and G8 only. In the diabetic + GYY 4137 group, the values at week 2 varied from G15 to G180 and reduced at week 4 between G60 and G100. The threshold was around G60. Overall, the data confirm a threshold value at G60 for all groups except the untreated diabetic group in which the threshold was between G4 and G8. FIGURE 2. Open in new tabDownload slide (A) Bar graph showing the results of hot plate analysis between all the experimental groups at week 2 and week 4 interval. (*p < 0.02) diabetic versus control, control + GYY 4137, and diabetic + GYY 4137 groups. (**p < 0.001) diabetic + GYY4137 versus diabetic group. (B) Bar graph showing the results of paw pressure analysis of the left hind limb between all the experimental groups at week 2 and week 4 intervals. At week 2, (*p < 0.017) diabetic group versus control group; (**p < 0.001) diabetic + GYY4137 group versus control group. At week 4, (*** p < 0.039) control groups versus diabetic group; (# p < 0.001) diabetic + GYY4137 group versus diabetic group. (C) Bar graph showing the results of paw pressure analysis of the right hind limb between all experimental groups at week 2 and week 4 intervals. At week 2, (*p < 0.009) diabetic group versus control groups; (**p < 0.002) diabetic + GYY 4137 group versus control groups. At week 4, (***p < 0.045) control group versus diabetic group; (****p < 0.001) diabetic + GYY 4137 group versus diabetic group. (D) Bar graph showing the results of Von Frey analysis between all the groups at week 2 and week 4 intervals. At week 4, (*p < 0.001) diabetic group versus control group; (**p < 0.05) diabetic + GYY 4137 group versus diabetic group. (E) Geometry graph showing the results of Von Frey analysis. The figure shows von Frey Test values for 5 animals each at weeks 2 and 4, for control, control with GYY4137, diabetic and diabetic with GYY4137 groups. In the controls, the values at week 2 varied from G10 to G180, and at week 4 from G10 to G60. The threshold was around G60. In the controls + GYY4137 group, the values at week 2 varied from G60 to G180, while at week 4 all the values were constant at G60. Hence, the threshold was good at G60. In the diabetic group, the values at week 2 varied from G6 to G60, and at week 4, the values declined to a level between G4 and G8 only. In the diabetic + GYY 4137 group, the values at week 2 varied from G15 to G180 and reduced at week 4 between G60 and G100. The threshold was around G60. Overall, the data confirm a threshold value at G60 for all groups except the untreated diabetic group in which the threshold was between G4 and G8. FIGURE 2. Open in new tabDownload slide continued FIGURE 2. Open in new tabDownload slide continued Paw Pressure Test The paw withdrawal reflex threshold of the right hind limb (Fig. 2B) showed a significant (p < 0.009) decrease in the STZ-induced diabetic animals compared with control and the GYY4137-treated groups. GYY4137-treated diabetic rats significantly (p < 0.045) improved with time compared with diabetic rats. Moreover, GYY4137-treated diabetic rats did not show any significant difference compared with control rats during week 2. However, in the fourth week, the paw withdrawal reflex threshold of diabetes-induced rats was significantly (p < 0.002) decreased than second week in the right hind limb compared with control groups. Also, GYY4137-treated diabetic rats showed a great significant (p < 0.001) difference compared with diabetic rats during the fourth week. Moreover, GYY4137-treated diabetic rats did not show any significant difference compared with control groups in the fourth week. Likewise, the diabetic animals showed a significant decrease in the paw withdrawal reflex threshold in the left hind limb in the second (p < 0.017) and fourth (p < 0.001) weeks of the experiment (Fig. 2C) compared with control groups. In contrast, the GYY4137-treated animals showed a significant increase in the paw withdrawal reflex threshold in week 2 (p < 0.039) and week 4 (p < 0.001) compared with the diabetic group. Further, GYY4137-treated diabetic rats showed a significant difference compared with control groups during week 4, but not in week 2. Von Frey Test The mean von Frey filament force (g ± SD) required inducing an FRT (static allodynia) was calculated and then graphed for the 4 groups (Fig. 2D). Analysis of the von Frey data did not reveal a significant difference in the FRT force expressed in the 4 experimental groups during the second week. However, the results showed a substantial difference between the 4 groups during the fourth week. Data revealed a significant (p < 0.001) reduction in the FRT force expressed in the diabetic group compared with the control groups. This decrease was significantly (p < 0.001) reversed in the GYY4137-treated diabetic animals at week 4 compared with the diabetic group. On the other hand, there was no significant difference between the GYY4137-treated diabetic group and the control groups. The geometry graph (Fig. 2E) shows the results of Von Frey analysis. The figure shows von Frey test values for the experimental animals each at weeks 2 and 4. In the control groups, the values at week 2 varied from G10 to G180, and at week 4 from G10 to G60. The threshold was around G60. In control + GYY4137 group, the values at week 2 varied from G60 to G180, while at week 4 all the values were constant at G60. Hence, the threshold was good at G60. The values of the diabetic group at week 2 varied from G6 to G60, whereas, the values declined to a level between G4 and G8 at week 4. However, the values of the diabetic + GYY4137 varied from G15 to G180 at week 2 and reduced between G60 and G100 at week 4. The threshold averaged around G60. Overall, the data confirm a threshold value at G60 for all groups except the untreated diabetic group in which their threshold was between G4 and G8. Assessment of Microglia, Astrocytes, and Neurons in the Spinal Cord Microglia Iba-1, a microglia-specific activation marker, immunohistochemical staining in the right (Fig. 3A) and left (Fig. 3B) dorsal gray horns of the spinal cord showed weak immunostaining in the control groups. However, the diabetic animals showed marked and strong Iba-1 immunostaining compared with control groups, whereas GYY4137 treatment of diabetic animals showed a remarkable decrease in Iba-1 immunostaining in both right and left dorsal gray horns of the spinal cord compared with the diabetic group. Similar results were obtained from the white matter columns of the spinal cord (data not shown). Morphometric analysis of the immunohistochemical study (Fig. 3C) revealed a significant (p < 0.00) increase in the number of Iba-1-immunostained microglia in both gray and white horns of the spinal cord in diabetic animals compared with control groups. In contrast, the number of the Iba-1-positive microglia in the gray and white matter of the spinal cord was significantly (p < 0.00) decreased in the GYY4137-treated diabetic animals compared with untreated diabetic group (Fig. 3C). Further, no significant difference in the number of Iba-1-immunostained microglia was found between the GYY4137-treated animals and the control groups in white or gray matter of the spinal cord. The Western blot densitometry analysis of the expression of the Iba-1 protein showed a significant (p < 0.012) increase in the diabetic groups compared with control groups. However, the GYY4137-treated diabetic animals showed a significant (p < 0.015) decrease in Iba-1 protein expression compared with the untreated diabetic group (Fig. 3D). Also, the results show that there is no significant difference between the Iba-1 protein concentration of control groups and the GYY4137-treated diabetic group. FIGURE 3. Open in new tabDownload slide (A) Photographs showing the Iba-1-immunoreactive microglia cells of the right dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a higher number of Iba-1-immunoreactive microglia cells compared with the control group. The GYY4137-treated diabetic group showed a remarkably decreasing number of immunoreactive microglia cells compared with the diabetic group. (B) Photographs comparing the Iba-1-immunoreactive microglia cells of the left dorsal gray matter of the spinal cord from the different experimental groups at different magnifications (40×, 100×, and 400×). Likewise, the diabetic group showed higher immunoreactive microglia cells compared with control groups. The GYY4137-treated diabetic group showed a decrease in the number of immunoreactive microglia cells compared with the diabetic group. (C) Graph comparing the number of Iba-1-immunoreactive microglia cells in the white and gray matter of the spinal cord of the experimental groups. The diabetic group showed significantly (*p < 0.0001) higher number of immunoreactive microglia cells in both white and gray matter compared with control groups. The GYY4137-treated diabetic group showed significant (**p < 0.001) decrease the number of microglial cells in the whole spinal cord compared with the diabetic group. (D) Western blot analysis of Iba-1 protein expression showing a significant increase of Iba-1 protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p value < 0.012; diabetic group versus control groups. **p < 0.015; GYY4137-treated diabetic group versus diabetic group. Values are mean ± SE; n = 6. FIGURE 3. Open in new tabDownload slide (A) Photographs showing the Iba-1-immunoreactive microglia cells of the right dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a higher number of Iba-1-immunoreactive microglia cells compared with the control group. The GYY4137-treated diabetic group showed a remarkably decreasing number of immunoreactive microglia cells compared with the diabetic group. (B) Photographs comparing the Iba-1-immunoreactive microglia cells of the left dorsal gray matter of the spinal cord from the different experimental groups at different magnifications (40×, 100×, and 400×). Likewise, the diabetic group showed higher immunoreactive microglia cells compared with control groups. The GYY4137-treated diabetic group showed a decrease in the number of immunoreactive microglia cells compared with the diabetic group. (C) Graph comparing the number of Iba-1-immunoreactive microglia cells in the white and gray matter of the spinal cord of the experimental groups. The diabetic group showed significantly (*p < 0.0001) higher number of immunoreactive microglia cells in both white and gray matter compared with control groups. The GYY4137-treated diabetic group showed significant (**p < 0.001) decrease the number of microglial cells in the whole spinal cord compared with the diabetic group. (D) Western blot analysis of Iba-1 protein expression showing a significant increase of Iba-1 protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p value < 0.012; diabetic group versus control groups. **p < 0.015; GYY4137-treated diabetic group versus diabetic group. Values are mean ± SE; n = 6. FIGURE 3. Open in new tabDownload slide continued FIGURE 3. Open in new tabDownload slide continued FIGURE 3. Open in new tabDownload slide continued FIGURE 3. Open in new tabDownload slide continued Astrocytes The GFAP astrocyte-specific activation marker immunohistochemical staining in the right (Fig. 4A) and left (Fig. 4B) dorsal gray horns of the spinal cord showed strong GFAP-immunostained astrocytes in the control groups. The diabetic animals showed a marked decrease in GFAP-immunostained astrocytes compared with control groups. However, GYY4137 treatment of diabetic animals showed a remarkable increase in the GFAP immunostaining in both right and left dorsal gray horns of the spinal cord compared with the diabetic group. Similar results were obtained from the white matter columns of the spinal cord (data not shown). Morphometric analysis of the immunohistochemical study (Fig. 4C) revealed a significant (p < 0.00) decrease in the number of GFAP-immunostained astrocytes in both gray and white matter of the spinal cord in diabetic animals compared with control groups. In contrast, the number of the GFAP-immunopositive astrocytes in the gray and white matter of the spinal cord was significantly (p < 0.001) increased in the GYY4137-treated diabetic animals compared with untreated diabetic group (Fig. 4C). Also, there was no significant difference in the number of GFAP-immunostained astrocytes between the GYY4137-treated animals and the control groups in white or gray matter of the spinal cord. The GFAP protein expression analysis of the spinal cord samples showed a significant (p < 0.011) decrease in the diabetic groups compared with control groups. However, the GYY4137-treated diabetic animals showed a significant (p < 0.021) increase in GFAP protein expression compared with the untreated diabetic group (Fig. 4D). The results also showed no significant difference between the GFAP protein concentration of the control groups and the GYY4137-treated diabetic group. FIGURE 4. Open in new tabDownload slide (A) Photographs showing the GFAP-immunoreactive astrocyte cells of the right dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a significantly lower number of GFAP-immunoreactive astrocyte cells compared with the control group. GYY4137-treated diabetic group showed a remarkable increase in the GFAP-immunoreactive astrocytes compared with the diabetic group. (B) Photographs showing the GFAP-immunoreactive astrocyte cells of the left dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a lower number of GFAP-immunoreactive astrocytes compared with the control group. In contrast, the spinal cord tissue of GYY4137-treated diabetic group showed an increase in the number of immunoreactive astrocytes compared with the diabetic group. (C) Graph comparing the number of GFAP-immunoreactive astrocytes in the white and gray matter areas of the spinal cord of the experimental groups. The diabetic group showed significantly (*p < 0.001) lower number of immunoreactive astrocyte cells compared with control groups (*p < 0.001). GYY4137-treated diabetic group showed a significant (*p < 0.001) increase in the number of GFAP-immunoreactive astrocytes compared with the diabetic group. Values are mean ± SE, n = 6. (D) Western blot analysis of GFAP protein expression showing a significant increase of GFAP protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p value < 0.012; diabetic group versus control groups. *p value < 0.016; diabetic group versus control groups. **p value < 0.005; GYY4137-treated group versus diabetic group. Values are mean ± SE, n = 6. FIGURE 4. Open in new tabDownload slide (A) Photographs showing the GFAP-immunoreactive astrocyte cells of the right dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a significantly lower number of GFAP-immunoreactive astrocyte cells compared with the control group. GYY4137-treated diabetic group showed a remarkable increase in the GFAP-immunoreactive astrocytes compared with the diabetic group. (B) Photographs showing the GFAP-immunoreactive astrocyte cells of the left dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a lower number of GFAP-immunoreactive astrocytes compared with the control group. In contrast, the spinal cord tissue of GYY4137-treated diabetic group showed an increase in the number of immunoreactive astrocytes compared with the diabetic group. (C) Graph comparing the number of GFAP-immunoreactive astrocytes in the white and gray matter areas of the spinal cord of the experimental groups. The diabetic group showed significantly (*p < 0.001) lower number of immunoreactive astrocyte cells compared with control groups (*p < 0.001). GYY4137-treated diabetic group showed a significant (*p < 0.001) increase in the number of GFAP-immunoreactive astrocytes compared with the diabetic group. Values are mean ± SE, n = 6. (D) Western blot analysis of GFAP protein expression showing a significant increase of GFAP protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p value < 0.012; diabetic group versus control groups. *p value < 0.016; diabetic group versus control groups. **p value < 0.005; GYY4137-treated group versus diabetic group. Values are mean ± SE, n = 6. FIGURE 4. Open in new tabDownload slide continued FIGURE 4. Open in new tabDownload slide continued FIGURE 4. Open in new tabDownload slide continued FIGURE 4. Open in new tabDownload slide continued Neurons The right (Fig. 5A), and left (Fig. 5B) dorsal gray horns of the spinal cord showed robust NeuN-immunostained neurons in the control groups whereas the dorsal gray horns of the diabetic animals showed a marked decrease in NeuN-immunostained neurons compared with control groups. However, the GYY4137-treated diabetic animals showed a remarkable increase in the NeuN immunostaining in both right and left dorsal gray horns of the spinal cord compared with the diabetic group. Morphometric analysis of the immunohistochemical study (Fig. 5C) revealed a significant (p < 0.00) decrease in the number of NeuN-immunostained neurons in the gray matter of the spinal cord in diabetic animals compared with control groups. In contrast, the number of the NeuN-immunopositive neurons in the right and left gray matter of the spinal cord was significantly (p < 0.00) increased in the GYY4137-treated diabetic animals compared with untreated diabetic group (Fig. 5C). Also, there was no significant difference in the number of NeuN-immunostained neurons between the GYY4137-treated animals and the control groups in the gray matter of the spinal cord. The NeuN protein expression analysis of the spinal cord samples showed a significant (p < 0.01) decrease in the diabetic groups compared with control groups. However, the GYY4137-treated diabetic animals showed a significant (p < 0.021) increase in GFAP protein expression compared with the untreated diabetic group (Fig. 4D). The results also showed no significant difference between the NeuN protein concentration of the control groups and the GYY4137-treated diabetic group. FIGURE 5. Open in new tabDownload slide (A) Photographs showing NeuN-immunoreactive neurons in the right dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a remarkable lower number of NeuN-immunoreactive neurons compared with the control group. GYY 4137-treated diabetic group showed an increase in the number of NeuN-immunoreactive neurons compared with the diabetic group. (B) Photographs showing NeuN-immunoreactive neurons in the left dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a remarkable lower number of NeuN-immunoreactive neurons compared with the control group. In contrast, GYY 4137-treated diabetic group showed an increasing number of immunoreactive neurons compared with the diabetic group. (C) Graph comparing the number of NeuN-immunoreactive neurons in the dorsal gray matter area of the spinal cord of the experimental groups. The diabetic group showed significantly (*p < 0.001) lower number of immunoreactive neurons compared with control groups. In contrast, GYY 4137-treated diabetic group showed a significant (**p < 0.001) increase in the number of NeuN-immunoreactive neurons compared with the diabetic group. (D) Western blot analysis of GFAP protein expression showing a significant increase of GFAP protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p value < 0.012; diabetic group versus control groups. *p value < 0.016; diabetic group versus control groups. *p value < 0.011; diabetic group versus control groups. **p value < 0.021; GYY 4137-treated diabetic group versus diabetic group. Values are mean ± SE, n = 6. FIGURE 5. Open in new tabDownload slide (A) Photographs showing NeuN-immunoreactive neurons in the right dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a remarkable lower number of NeuN-immunoreactive neurons compared with the control group. GYY 4137-treated diabetic group showed an increase in the number of NeuN-immunoreactive neurons compared with the diabetic group. (B) Photographs showing NeuN-immunoreactive neurons in the left dorsal gray matter of the spinal cord in the different experimental groups at different magnifications (40×, 100×, and 400×). The diabetic group showed a remarkable lower number of NeuN-immunoreactive neurons compared with the control group. In contrast, GYY 4137-treated diabetic group showed an increasing number of immunoreactive neurons compared with the diabetic group. (C) Graph comparing the number of NeuN-immunoreactive neurons in the dorsal gray matter area of the spinal cord of the experimental groups. The diabetic group showed significantly (*p < 0.001) lower number of immunoreactive neurons compared with control groups. In contrast, GYY 4137-treated diabetic group showed a significant (**p < 0.001) increase in the number of NeuN-immunoreactive neurons compared with the diabetic group. (D) Western blot analysis of GFAP protein expression showing a significant increase of GFAP protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p value < 0.012; diabetic group versus control groups. *p value < 0.016; diabetic group versus control groups. *p value < 0.011; diabetic group versus control groups. **p value < 0.021; GYY 4137-treated diabetic group versus diabetic group. Values are mean ± SE, n = 6. FIGURE 5. Open in new tabDownload slide continued FIGURE 5. Open in new tabDownload slide continued FIGURE 5. Open in new tabDownload slide continued FIGURE 5. Open in new tabDownload slide continued IL1-β Immunoblotting IL1-β protein immunoblot (band size is ∼17 kDa) and densitometric analysis (Fig. 6A) showed a significant (p < 0.016) increase in diabetic animals compared with control groups. The GYY4137 treatment of the diabetic animals showed a significant (p < 0.005) decrease in the expression of the IL1-β protein compared with the untreated diabetic group. Also, the results show no significant difference between the IL-1β protein concentration of control groups and the GYY4137-treated diabetic group. FIGURE 6. Open in new tabDownload slide Western immunoblot protein analysis of L1-β, IL-6, and TNF-α in the spinal cords of the 4 experimental groups at the end of the experiments. (A) Densitometry analysis of IL-1β protein expression showed a significant increase of IL-1β protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p < 0.016; diabetic group versus control groups. **p < 0.005; GYY 4137-treated group versus diabetic group. Values are mean ± SE, n = 6. (B) IL-6 protein expression analysis showing a significant increase of IL-6 protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with the diabetic group. *p < 0.001; diabetic group versus control groups. **p < 0.004; GYY 4137-treated diabetic group versus diabetic group. Values are mean ± SE, n = 6. (C) Densitometry of TNF-α protein band expression. Western blot analysis of TNF-α protein expression showing a significant increase of TNF-α protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p ≤ 0.011; diabetic group versus control groups. **p < 0.021; GYY 4137-treated diabetic group versus diabetic group. Values are mean ± SE; n = 6. FIGURE 6. Open in new tabDownload slide Western immunoblot protein analysis of L1-β, IL-6, and TNF-α in the spinal cords of the 4 experimental groups at the end of the experiments. (A) Densitometry analysis of IL-1β protein expression showed a significant increase of IL-1β protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p < 0.016; diabetic group versus control groups. **p < 0.005; GYY 4137-treated group versus diabetic group. Values are mean ± SE, n = 6. (B) IL-6 protein expression analysis showing a significant increase of IL-6 protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with the diabetic group. *p < 0.001; diabetic group versus control groups. **p < 0.004; GYY 4137-treated diabetic group versus diabetic group. Values are mean ± SE, n = 6. (C) Densitometry of TNF-α protein band expression. Western blot analysis of TNF-α protein expression showing a significant increase of TNF-α protein density in the spinal cord of diabetic group compared with control animals; whereas, diabetic GYY4137-treated animals showed a significant decrease compared with diabetic group. *p ≤ 0.011; diabetic group versus control groups. **p < 0.021; GYY 4137-treated diabetic group versus diabetic group. Values are mean ± SE; n = 6. IL-6 Immunoblotting Diabetic animals showed a significant (p < 0.001) increase in the IL-6 protein expression (band size is ∼21 kDa) compared with control groups (Fig. 6B). The expression of the IL-6 protein was almost similar between the control + GYY4137 and diabetic + GYY 4137 groups. The IL-6 protein expression was significantly (p < 0.004) reduced in the GYY4137-treated diabetic animals compared with the untreated diabetic group. The data also show that there is no significant difference between IL-6 protein concentration of the controls and GYY4137-treated diabetic groups. TNF-α Immunoblotting TNF-α protein densitometry (band size is ∼17 kDa) analysis results are shown in (Fig. 6C). The diabetic animals showed a significant (p < 0.011) increase in the TNF-α protein expression compared with control and control + GYY4137 groups. The TNF-α protein expression was significantly (p < 0.021) reduced in the diabetic + GYY4137-treated animals compared with the untreated diabetic group. Also, the results show that there was no significant difference between TNF-α protein concentration of control groups and the GYY4137-treated diabetic group. DISCUSSION Significant and rapid progress has been made in the study of biogases, such as H2S (34). H2S has become the focus of research due to its antioxidative, anti-inflammatory, and anti-apoptotic properties (35–37). Protective effects of H2S donors have been demonstrated against hyperglycemic injury in microvascular endothelial cells (38), myocardial ischemia (39), hypertension (40), cancer (41), osteoarthritis (42), gastrointestinal disorders (43), in addition to suppression of diabetes-accelerated atherosclerosis (44). Also, its anti-inflammatory and cytoprotective properties have been documented. H2S alleviates the development of diabetic cardiomyopathy, diabetic nephropathy, diabetic retinopathy via attenuation of inflammation, oxidative stress, and apoptosis (19, 45). The current study aimed to emphasize the effects of slow-releasing H2S donor GYY4137 treatment on the reduction of allodynia and hyperalgesia as well as microglial activation and the changes in the production of proinflammatory cytokines in diabetic rats. GYY4137-treated STZ-induced diabetic rats showed increased body weight gain and hyperglycemia, indicating a crucial role of H2S in fat metabolism. Several studies have reported that H2S has a vital role in adipogenesis and fat mass accumulation through the enhancement of adipocytes function. Moreover, it was suggested that H2S is involved in the pathogenesis of obesity in mice (46). H2S is synthesized in the adipose tissue mainly by cystathionine γ-lyase. Further, H2S has arisen as an essential factor in the modulation of insulin action in adipose tissue. Tsai et al examined the role of H2S in the regulation of adipogenesis using a fibroblast-like mouse preadipocyte cell line (3T3-L1) and reported that H2S modulates adipogenesis and adipocyte maturation (47). Similarly, Cai and collaborators demonstrated that GYY4137 increased lipid accumulation in these cells (48). GYY4137 inhibited lipolysis in mice in vivo without increasing fat mass (49). Similarly, we found that the weight of H2S-treated diabetic rats increased in week 4 when compared with the diabetic group in the same week. Also, there was a significant difference between the weight of GYY4137-treated and untreated diabetic rats in the fourth week, which may indicate that H2S has a vital role in adipogenesis. GYY4137 likely protects against high glucose-induced cytotoxicity in H9c2 cells (50). In mice, GYY4137 increases plasma glucose and insulin concentrations associated with impaired insulin sensitivity (49). However, our results did not show any beneficial effect of GYY4137 on glucose levels, as reported earlier by Szabo (51). This may be attributed to the side effect of H2S, which can cause damage to insulin-producing Beta cells in the pancreas (51). Moreover, the weight of the spinal cord of diabetic rats was significantly higher when compared with other groups. This finding does not match with the body weight decrease in diabetic rats. Whether the activated microglia are a cause or a consequence of weight loss is unclear. It needs more investigations and may be due to the activation of microglial cells and the recruitment of other cells. Antiallodynic/Antihyperalgesic Effect of H2S on STZ-Induced Diabetic Rats Neuropathic pain is characterized by sensory abnormalities, such as unpleasant abnormal sensation (dysesthesia), an increased response to mechanical nociceptive stimuli (hyperalgesia) and in response to a stimulus that does not normally provoke pain (allodynia). Our study showed that STZ-induced diabetic rats displayed mechanical hyperalgesia and allodynia, similar to clinical symptoms in DM patients (52). The behavioral alterations started on the second week after STZ-induced diabetes and lasted through the experimental period. However, H2S treatment significantly inhibited STZ-induced mechanical allodynia and mechanical hyperalgesia. These observations are in line with earlier findings (53). The increase in nociception thresholds in untreated diabetic rats indicates a loss of nociceptive perception that could be attributed to nerve damage caused by the development of diabetic neuropathy (54). On the other hand, STZ-induced diabetic rats showed an increase in thermal (hot plate) latency time. This increase suggests the presence of thermal nociception in diabetic animals. In diabetic neuropathy, the damage of peripheral nerves may result in loss of pain perception (54, 55). In the present study, the pain threshold measured by hot plate increased significantly compared with control groups indicated the loss of pain perception (54). The GYY4137 treatment attenuated the thermal (hot plate) reflex latency time to the levels comparable to control groups. This attenuation indicates the effectiveness of H2S therapy in alleviating the thermal antinociceptive effect seen in diabetic subjects. Interestingly, it seems that the H2S treatment acts on oppositely by increasing the allodynia and mechanical hyperalgesia latencies while decreasing the thermal threshold by working on different mechanical and thermal nociceptors selectively. This remains to be further investigated. Noxious stimulation evokes changes in many systems within the CNS. In addition to the paw withdrawal reflexes, reactions, such as licking, escaping, and head orientation, are also recognized as supraspinal indicators. Sensory signals are transmitted from the periphery by primary afferent fibers into the dorsal horn, where these afferents synapse with intrinsic spinal dorsal horn neurons. Central afferent fibers terminate superficially in laminae I–II, with a smaller number reaching deeper laminae (56). In contrast, Ab-fibers predominantly innervate laminae III–VI therefore respond to the full range of stimulation, from light touch to noxious pinch, heat, and chemicals (57). Spinal projection neurons then convey this information to supraspinal centers, where nonnoxious and noxious signals can be perceived. Spinal projection neurons carry the dorsal horn output to higher centers in the brain along ascending pathways and have been shown to project to the thalamus, limbic system, and various cortical areas (pain matrix) periaqueductal gray, rostral ventromedial medulla and specifically to the parabrachial area. In the CNS, the pain information is controlled by ascending and descending inhibitory/excitatory systems, using different neurotransmitters (56). The role of inflammatory mediators is not limited to peripheral components but may extend to the supraspinal centers, starting in the dorsal horn of the spinal cord (58). Early studies have shown a vital role for the increased level of IL-6 in allodynia and hyperalgesia in a rat model of mononeuropathy (59), the activation of microglia following nerve fiber injury (60), the contribution of this activation to the development of tactile allodynia (61) and the possible attenuation of allodynia and hyperalgesia by preventive treatment with an inhibitor of microglial activation (62). Sustained and significant changes in the behavior of central neurons induced by peripheral nerve injuries have been described in brainstem centers, the thalamus, the hypothalamus, and the cerebral cortical and subcortical areas involved in the processing of nociception (58). Thus, all of these manifestations suggest plastic changes at various supraspinal levels induced by injury or trauma to peripheral nerve fibers. Therefore, the supraspinal pain-modulation system that is conveyed by the descending inhibitory fibers modulate (block) the input and output of laminae I, II, V, and VII spinal neurons. In this regard, it was also shown that supraspinal µ-opioid receptors were responsible for the antinociceptive effects of fentanyl (i.p. or i.t. doses) in diabetes-induced neuropathic pain (63). Further, the induction of neuropathic pain by STZ-induced diabetes renders spinal cord µ-opioid systems ineffective in producing antinociception for noxious heat, electrical, and pressure stimuli (64). Following peripheral diabetic neuropathy or conditions of inflammation, changes can occur in these excitatory and inhibitory processes that modulate spinal excitability, often resulting in the heightened response of dorsal neurons to incoming afferent signals increased output to the brain, a phenomenon known as central sensitization. An earlier study demonstrated a strong inhibitory effect of dorsal column stimulation on neuropathic pain. This inhibition was attributed to brainstem pain-modulating centers' activation via rostral projections of the dorsal column nuclei (64). In the light of earlier studies, GYY4137 treatment may act as an analgesic drug and anti-inflammatory modulator on the supraspinal centers and the spinal cord neurons. The decrease in the number of NeuN-reactive spinal neurons in the diabetic animals may indicate severe damage to the spinal interneurons and projection spinothalamic pathway neurons (discriminatory sensory aspects of nociception) and spino-parabrachial pathways to the supraspinal centers (affective reactions to pain mechanisms and in regulating descending control systems) that in turn will affect the induction of pain. The pathological changes seen in the spinal astrocytes and microglia may also exaggerate the damaging effect on both spinothalamic and spino-parabrachial pathways. Further studies need to be done to assess the histopathological, physiological, and pharmacological supraspinal changes following GYY4137 treatment of diabetic-induced peripheral neuropathy. Effect of H2S on Spinal Microglial Activation in STZ-Induced Diabetic Rats Increasing evidence demonstrates that activated microglia in the spinal cord contributes to the development of neuropathic pain in diabetes (17). Microglia are innate immune cells that initiate an inflammatory response by secreting an excessive array of inflammatory mediators that activate other immune cells, then launching a consecutive inflammatory sequence. Further, microglia are crucial cellular mediators of plasticity in the spinal cord that determine the development and maintenance of pain hypersensitivity following peripheral nerve damage (36). Expression of Iba1, expressed explicitly in microglia, is upregulated upon activation of microglia due to inflammation, allowing the differentiation between surveilling and activated microglia. In the present study, the GYY4137 treatment of the diabetic animals completely attenuated the increase of Iba-1 protein expression in diabetic animals at week 2 and week 4. Further, recent data have shown that the inhibition of microglial activation prevents the development of neuropathic pain behavior in animal models of neuropathic pain (65). Recent studies have strongly supported the concept that spinal cord glia (including astrocytes and microglia) and proinflammatory cytokines, such as IL-1β, are involved in the induction and maintenance of neuropathic pain (66). Diabetic complications implicate a subset of cell types, such as capillary endothelial cells, mesangial cells, neurons, and Schwann cells (in peripheral nerves) that should benefit from H2S supplementation (38). In STZ-induced diabetic rats, microglia are activated and start to proliferate and change their morphology from ramified state to ameboid state (36, 67). The number of microglia increased in the spinal cord of STZ-induced diabetic rats, proposing that proliferation or migration is enhanced (68). In this study, we report that H2S treatment decreased the number of activated microglia cells to baseline levels compared with the significantly increased number of microglia in the spinal cord of diabetic rats. Activated microglia at the site of inflammation modify many of their molecular and morphological properties (69) and express increased levels of MHC antigens and became phagocytic (70). Microglia release inflammatory cytokines that initiate the inflammatory reaction cascade by recruiting and activating other cells to the CNS lesion. Also, they can release potent proapoptotic cytokines, such as TNF-α and IL-1β, which can cause neuronal damage. The expression of proinflammatory cytokines was induced and resulted in M1 microglial activation in the young offspring rats after MSD (71). Therefore, ceasing the deliverance of proinflammatory mediators from microglia is one approach to alleviate constant inflammatory conditions. Hence, the intervention of the microglial activation process will become a promising therapeutic target for the treatment of diabetic neuropathy. H2S Downregulates Proinflammatory Cytokines IL-1β, IL 6, and TNF-α, in the Spinal Cord of STZ-Induced Diabetic Rats One of the likely underlying mechanisms of neuropathic pain is neuroinflammation. Following diabetes, the immune system becomes activated both in the periphery and CNS. The development of neuroinflammation is based on the activation of inflammatory and immune-like glial cells at the peripheral level and microglia and astrocytes in the CNS (72). Since proinflammatory cytokines are critical in the initiation and maintenance of neuropathic pain, we investigated the effect of H2S on inhibiting inflammation caused by diabetes through their downregulation. GYY4137 treatment exhibited anti-inflammatory effects in the spinal cord of STZ-treated rats. Similarly, in STZ-induced diabetic neuropathy there are increased proinflammatory cytokines, such as IL-6 mRNA levels in both the DRG and sciatic nerve compared with nondiabetic mice (28). GYY4137 decomposition products appear inactive, allowing the researcher to study the physiological effects of the slow release of H2S in inflammation directly in the absence of any possible effects (73). Investigating the impact of slow-release H2S using GYY4137 has exclusively shown in several cellular and in vivo animal models that the slow-release of H2S is anti-inflammatory (73). GYY4137 demonstrated anti-inflammation activity in vivo (73), antioxidant, and anti-apoptotic effects (74). The upregulation of IL-1β is a common constituent in chronic pain (75). IL-1β is an essential component in the nervous system and is considered a potent inflammatory cytokine involved in many critical cellular functions, such as proliferation, cell activation, and differentiation. The release of IL-1β constitutes an essential step in the inflammation process, which is achieved through the induction of other inflammatory cytokines, such as IL-6 and TNF-α (76). Our results show a remarkable attenuation of in IL-1β expression diabetic animals following H2S treatment down to normal levels shown similar to controls animals. Moreover, the expression of IL-6 protein and TNF-α were highly increased in diabetic groups as compared with the control group. We showed that GYY4137, downregulates the secretion of these inflammatory mediators from STZ-induced rats at doses that are nontoxic to the cells, thereby demonstrating its efficacy in vivo. Our data agree with a recent study in which H2S donor treatment reduced cytokine production and exhibited anti-inflammatory activity in vivo by decreasing plasma TNF-α, IL-1β, and IL-6 levels following lipopolysaccharide challenge (77). IL-6 has been involved as a key mediator in the development of neuropathic pain behavior (78). For example, Schoeniger-Skinner et al reported that intrathecal administration of IL-6 mediates low-threshold mechanical allodynia in rats (79). The relation between IL-6 and neuropathic pain behavior seems to be consistent with our results that inhibition of IL-6 in the spinal cord by GYY4137 resulted in inhibiting the progressing of neuropathic pain behavior. A recent study showed that the upregulation of IL-6, and TNF-α in activated microglia was strongly inhibited in primary microglial cell culture by H2S (80). In inflammation, microglia may worsen its effect and cause neuronal and astrocytic glial degeneration through enhancing the expression of IL-1β, TNF-α, IL-6 proteins, and pain transmission in diabetic rats. H2S shows the significant inhibitory effect of spinal microglial activation and proliferation, which consequently lower the expression of proinflammatory cytokines and attenuating the progression of STZ-induced diabetes-related neuropathic pain. However, more work needs to be done to further investigate how spinal microglia are deactivated following H2S treatment and how they cause signaling to neurons, possibly other glial cells, in the dorsal horn pain transmission network. We observed a selective decrease in the number of GFAP-immunoreactive astrocytes in the spinal cords of STZ-induced diabetic rats, as previously reported (81). Concomitantly, diabetic animals showed a significant decrease in the number of NeuN-immunoreactive neurons. The GYY4137 treatment remarkably alleviated the reduction in the number of astrocytic and neuronal immunoreactive cells. The decline of the number of GFAP-immunoreactive astrocytes is comparable to other reports of significant reductions in GFAP levels in the hippocampus, cerebellum, white matter regions of the corpus callosum and external capsule (82) and retinas (83) of STZ-induced diabetic rats and BB/Wor diabetes-prone rats (84). Also, Goodison et al reported a reduction in GFAP mRNA expression in the cerebral cortex of Alzheimer disease and Down syndrome subjects (85). Likewise, Dennis et al showed that GFAP content and immunoreactivity were reduced in the sensory olfactory epithelium and olfactory bulb of 8 weeks STZ-diabetic animals compared with the nondiabetic controls (86). In addition to GFAP’s vital role as a cytoskeletal protein in which it provides structural stability of the astrocyte processes, many studies have examined the other possible functions of GFAP in the astrocytes of the CNS, myelination of CNS axons, blood-brain barrier (BBB), and synaptic transmission. Severe changes in the BBB, including increased permeability, along with changes in the CNS capillary density, accelerated aging of the vascular wall, and impaired vascular reactivity, which contributes to diabetes microangiopathy, have been reported in the CNS of diabetic patients and diabetic animal models (87, 88). Because astrocytes produce and secrete some important neurotrophic factors, the absence of GFAP or decrease in the astrocytes might interrupt this secretion, leading to neuropathy (89). Further, astrocytic processes are closely linked with the synaptic cleft, and many of these processes regulate synaptic functions by uptake of excess neurotransmitters, protecting neurons from reactive oxygen species (ROS), and producing some growth factors. In this regard, it was shown that GFAP is crucial for astrocyte-neuronal interactions in mice with a null mutation in the GFAP gene (89). These alterations were accompanied by poor vascularization of white matter and structural/functional changes in the BBB in older GFAP knockout mice (90). The decrease in the populations of GFAP-positive astrocytes and GFAP levels in diabetic animals is possibly linked to altered metabolic and functional capacities of the astrocytes and may, therefore, reduce their ability to maintain their roles in neuronal support in the CNS (91). A possible mechanism underlying the reduction in the immunoreaction of GFAP in diabetic animals is the absence or decrease in insulin levels. Several studies have reported that insulin is necessary to make GFAP staining highly intense in astrocytes’ cell bodies and processes and that it makes astrocytic processes more distinct and thicker (92). Because insulin is responsible for the increase in the expression of GFAP mRNA and peptide (93), it is likely that the absence of insulin could directly contribute to the downregulation of GFAP expression in the astrocytes. Insulin was reported to stimulate the production of GLUT-1 and GLUT-3 mRNA transporters through insulin receptors in astrocytes (94). The absence or reduction of insulin or insulin receptors seen in diabetes could change the intracellular metabolic processes in the astrocytes leading to the downregulation of GFAP expression in the spinal cord of diabetic rats, as seen in our study. Our current research shows a significant reduction in the GFAP immunoreactivity in the gray matter of the spinal cord of an STZ-induced diabetic model, a finding that agrees with the previously reported studies performed on the spinal cord (81) as well as other regions of the CNS, such as the brain and the retina. These results suggest that diabetes causes glial injury and alters glial behavior. Our findings may also be linked to altered metabolic activities and functional capacities of astrocytes that lead to a reduction in their ability to maintain the essential support and survival of the neurons in the spinal cord with consequent diabetic neuropathy. However, the GYY4137 treatment of diabetic rats was able to completely restore the reduction not only in the GFAP-immunostained astrocytes in gray and white matter but also in the NeuN-immunostained neurons of the spinal cord. These observations suggest that H2S confers protective effects on neuropathic pain in STZ-induced diabetes, possibly via inhibiting microglial activation and inflammation in the spinal cord leading to the protection of both astrocytes and neurons and restoring neurobehavioral sensory function in diabetic animals. STZ-induced diabetic animals also showed a significant reduction in the number of NeuN-immunoreactive neurons in the spinal cord. However, H2S treatments protected spinal neurons and maintained the standard neuronal number similar to nondiabetic groups. This protection indicates that GYY4137 may have an anti-apoptotic or antioxidative effect on the diabetic spinal neurons along with supporting glial cells. Several studies have shown that the state of hyperglycemia promotes apoptosis in various types of cells in diabetic neuropathy. In vivo and in vitro investigations confirmed that apoptosis is one of the potential mechanisms for hyperglycemia-induced neural cell death (95–97). Studies showed that hyperglycemia causes a neuronal cell oxidative injury through NADPH oxidase-dependent production of ROS (98). Besides, ROS generation through hyperglycemia promotes apoptosis, which is a probable mechanism of glucose neurotoxicity. Hyperglycemia activates numerous metabolic pathways like the polyol pathway, protein kinase C pathway, advanced glycation end products pathway, and hexosamine pathway. These pathways are shown to integrate through hyperglycemia mediated mitochondrial ROS production. Oxidative stress and these classical pathways in combination activate transcription factors, such as nuclear factor-kappa enhancer of B cells (NF-κB) and specialty protein-1 (SP-1), resulting in neuroinflammation and vascular impairment (95, 96, 98). Further, these pathways, combined with dysfunctional mitochondria-mediated apoptosis or bioenergetic depletion, can lead to neuronal damage leading to diabetic neuropathy (95–97). The crosstalk between oxidative stress and inflammation is due to the activation of NF-κ B and AP-1, inhibition of Nrf-2, peroxynitrite-mediated endothelial dysfunction, altered nitric oxide levels, and macrophage migration. All these mechanisms lead to the production of proinflammatory cytokines, which are responsible for nerve tissue damage and debilitating neuropathies (95). The Bcl-2 and Bax apoptotic genes also substantially contribute to diabetes and diabetic neuropathy. Bax was shown to be consistently strong to very strong, while Bcl-2 protein expression became weak to negative in diabetic retinopathy patients (99). The exact role of H2S modulation of apoptosis and oxidative stress processes needs to be further investigated to illustrate the changes in the neurons and supportive glial cells in diabetic neuropathy. In conclusion, the current study revealed the protective effects of H2S, which prevented neuropathic pain behavior in STZ-induced diabetic rats. The protective effects of H2S were associated with the prevention of microglial activation as well as attenuation of the upregulation of inflammatory cytokines in the spinal cord. Additionally, treatment with the GYY4137 protected the astrocytes and neurons of the spinal cord, which may be due to the decrease in the microglial activation and suppression of inflammatory cytokines. The ability of GYY4137 to attenuate the neuropathic pain behavior by preventing the development of mechanical allodynia, mechanical hyperalgesia and thermal antinociception in diabetic rats may be considered as a potential novel therapeutic approach of peripheral nerve neuropathy in diabetic patients. Therefore, these findings suggest that spinal microglial inhibition may hold a therapeutic promise in the treatment of DNP. This study was funded by the College of Graduate Studies and the Research Administration at Kuwait University (grant award Project No. YM02/17). The authors have no duality or conflicts of interest to declare. Supplementary Data can be found at https://academic.oup.com/jnen. ACKNOWLEDGMENTS We would like to express our sincere gratitude to Mrs. Vidya Alexander, Mrs. Monira Al-Mutawa, Mr. Jijin, and Mrs. Preethi George, for their technical assistance. Special thanks go to the Core Facility (OMICS RU Project # SRUL02/13) and Animal House Facility at Health Science Center, Kuwait University. REFERENCES 1 Shrestha SS , Zhang P , Albright A , et al. Medical expenditures associated with diabetes among privately insured US youth in 2007 . Diabetes Care 2011 ; 34 : 1097 – 101 Google Scholar Crossref Search ADS PubMed WorldCat 2 Baynes HW. Classification, pathophysiology, diagnosis and management of diabetes mellitus . J Diabetes Metab 2015 ; 6 : 541 . Google Scholar OpenURL Placeholder Text WorldCat 3 Biessels GJ , Bril V , Calcutt NA , et al. Phenotyping animal models of diabetic neuropathy: A consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab) . J Peripher Nerv Syst 2014 ; 19 : 77 – 87 Google Scholar Crossref Search ADS PubMed WorldCat 4 Bansal V , Kalita J , Misra UK. Diabetic neuropathy . Postgrad Med J 2006 ; 82 : 95 – 100 Google Scholar Crossref Search ADS PubMed WorldCat 5 Tesfaye S , Boulton AJ , Dyck PJ , et al. Diabetic neuropathies: Update on definitions, diagnostic criteria, estimation of severity, and treatments . Diabetes Care 2010 ; 33 : 2285 – 93 Google Scholar Crossref Search ADS PubMed WorldCat 6 Obrosova IG. Diabetic painful and insensate neuropathy: Pathogenesis and potential treatments . Neurotherapeutics 2009 ; 6 : 638 – 47 Google Scholar Crossref Search ADS PubMed WorldCat 7 Marchettini P , Lacerenza M , Mauri E , et al. Painful peripheral neuropathies . Curr Neuropharmacol 2006 ; 4 : 175 – 81 Google Scholar Crossref Search ADS PubMed WorldCat 8 Kriegstein A , Alvarez-Buylla A. The glial nature of embryonic and adult neural stem cells . Annu Rev Neurosci 2009 ; 32 : 149 – 84 Google Scholar Crossref Search ADS PubMed WorldCat 9 Jha MK , Jeon S , Su K. Glia as a link between neuroinflammation and neuropathic pain . Immune Netw 2012 ; 12 : 41 – 7 Google Scholar Crossref Search ADS PubMed WorldCat 10 Wang WY , Tan MS , Yu JT , et al. Role of proinflammatory cytokines released from microglia in Alzheimer's disease . Ann Transl Med 2015 ; 3 : 136 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 11 Peng J , Gu N , Zhou L , et al. Microglia and monocytes synergistically promote the transition from acute to chronic pain after nerve injury . Nat Commun 2016 ; 7 : 12029 Google Scholar Crossref Search ADS PubMed WorldCat 12 Tsuda M. Microglia in the spinal cord and neuropathic pain . J Diabetes Invest 2016 ; 7 : 17 – 26 Google Scholar Crossref Search ADS WorldCat 13 Streit WJ , Xue QS , Tischer J , et al. Microglial pathology . Acta Neuropathol Commun 2014 ; 2 : 142 Google Scholar Crossref Search ADS PubMed WorldCat 14 Boddeke EW. Involvement of chemokines in pain . Eur J Pharmacol 2001 ; 429 : 115 – 9 Google Scholar Crossref Search ADS PubMed WorldCat 15 Calvo M , Bennett DL. The mechanisms of microgliosis and pain following peripheral nerve injury . Exp Neurol 2012 ; 234 : 271 – 82 Google Scholar Crossref Search ADS PubMed WorldCat 16 Tennant F. Centralized pain: Our new clinical challenge . Pain 2013 ; 1 : 11 – 4 . Google Scholar OpenURL Placeholder Text WorldCat 17 Wang D , Couture R , Hong Y. Activated microglia in the spinal cord underlies diabetic neuropathic pain . Eur J Pharmacol 2014 ; 728 : 59 – 66 Google Scholar Crossref Search ADS PubMed WorldCat 18 Kimura Y , Dargusch R , Schubert D , et al. Hydrogen sulfide protects HT22 neuronal cells from oxidative stress . Antioxid Redox Signal 2006 ; 8 : 661 – 70 Google Scholar Crossref Search ADS PubMed WorldCat 19 Zhou X , Feng Y , Zhan Z , et al. Hydrogen sulfide alleviates diabetic nephropathy in a streptozotocin-induced diabetic rat model . J Biol Chem 2014 ; 289 : 28827 – 34 Google Scholar Crossref Search ADS PubMed WorldCat 20 Renno WM , Khan KM , Benov L. Is there a role for neurotrophic factors and their receptors in augmenting the neuroprotective effect of (–)-epigallocatechin-3-gallate treatment of sciatic nerve crush injury? Neuropharmacology 2016 ; 102 : 1 – 20 Google Scholar Crossref Search ADS PubMed WorldCat 21 Renno WM , Benov L , Khan KM. Possible role of antioxidative capacity of (–)-epigallocatechin-3-gallate treatment in morphological and neurobehavioral recovery after sciatic nerve crush injury . J Neurosurg Spine 2017 ; 27 : 593 – 613 Google Scholar Crossref Search ADS PubMed WorldCat 22 Li L , Whiteman M , Guan YY , et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): New insights into the biology of hydrogen sulfide . Circulation 2008 ; 117 : 2351 – 60 Google Scholar Crossref Search ADS PubMed WorldCat 23 Yousif MH , Dhaunsi GS , Makki BM , et al. Characterization of Angiotensin-(1–7) effects on the cardiovascular system in an experimental model of Type-1 diabetes . Pharmacological Research 2012 ; 66 : 269 – 75 Google Scholar Crossref Search ADS PubMed WorldCat 24 Qabazard B , Yousif MHM , Phillips OA. Alleviation of impaired reactivity in the corpus cavernosum of STZ-diabetic rats by slow-release H2S donor GYY4137 . Int J Impot Res 2019 ; 31 : 111 – 8 Google Scholar Crossref Search ADS PubMed WorldCat 25 Renno WM , Saleh F , Klepacek I , et al. Green tea pain modulating effect in sciatic nerve chronic constriction injury rat model . Nutr Neurosci 2006 ; 9 : 41 – 7 Google Scholar Crossref Search ADS PubMed WorldCat 26 Chaplan SR , Bach FW , Pogrel JW , et al. Quantitative assessment of tactile allodynia in the rat paw . J Neurosci Methods 1994 ; 53 : 55 – 63 Google Scholar Crossref Search ADS PubMed WorldCat 27 Christensen MD , Hulsebosch CE. Chronic central pain after spinal cord injury . J Neurotrauma 1997 ; 14 : 517 – 37 Google Scholar Crossref Search ADS PubMed WorldCat 28 Yoon H , Thakur V , Chattopadhyay M. Changes in inflammatory mediators in DRG of type 1 diabetic animals with neuropathy . Diabetes 2014 ; 63 : A149 . Google Scholar OpenURL Placeholder Text WorldCat 29 Al-Adwani DG , Renno WM , Orabi KY. Neurotherapeutic effects of ginkgo biloba extract and its terpene trilactone, ginkgolide B, on sciatic crush injury model: A new evidence . PLoS One 2019 ; 14 : e0226626 Google Scholar Crossref Search ADS PubMed WorldCat 30 Lal H , Fielding S. Psychopharmacology of clonidine: An introduction . Prog Clin Biol Res 1981 ; 71 : 1 – 3 Google Scholar PubMed OpenURL Placeholder Text WorldCat 31 Randall LO , Selitto JJ. Archives Internationales de Pharmacodynamie et de . Thérapie (Hashi Trust) 1957 ; 133 : 233 . Google Scholar OpenURL Placeholder Text WorldCat 32 Renno WM , Al-Maghrebi M , Rao MS , et al. (–)-Epigallocatechin-3-gallate modulates spinal cord neuronal degeneration by enhancing growth-associated protein 43, B-cell lymphoma 2, and decreasing B-cell lymphoma 2-associated x protein expression after sciatic nerve crush injury . J Neurotrauma 2015 ; 32 : 170 – 84 Google Scholar Crossref Search ADS PubMed WorldCat 33 Tseng TJ , Chen CC , Hsieh YL , et al. Influences of surgical decompression on the dorsal horn after chronic constriction injury: Changes in peptidergic and delta-opioid receptor (+) nerve terminals . Neuroscience 2008 ; 156 : 758 – 68 Google Scholar Crossref Search ADS PubMed WorldCat 34 Yang X , Hao D , Zhang H , et al. Treatment with hydrogen sulfide attenuates sublesional skeletal deterioration following motor complete spinal cord injury in rats . Osteoporos Int 2017 ; 28 : 687 – 95 Google Scholar Crossref Search ADS PubMed WorldCat 35 Xie K , Yu Y , Pei Y , et al. Protective effects of hydrogen gas on murine polymicrobial sepsis via reducing oxidative stress and HMGB1 release . Shock 2010 ; 34 : 90 – 7 Google Scholar Crossref Search ADS PubMed WorldCat 36 Zhao LL , Hu GC , Zhu SS , et al. Propofol pretreatment attenuates lipopolysaccharide-induced acute lung injury in rats by activating the phosphoinositide-3-kinase/Akt pathway . Braz J Med Biol Res 2014 ; 47 : 1062 – 7 Google Scholar Crossref Search ADS PubMed WorldCat 37 Chen X , Liu Q , Wang D , et al. Protective effects of hydrogen-rich saline on rats with smoke inhalation injury . Oxid Med Cell Longev 2015 ; 2015 : 106836 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 38 Gerő D , Torregrossa R , Perry A , et al. The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro . Pharmacol Res 2016 ; 113 : 186 – 98 Google Scholar Crossref Search ADS PubMed WorldCat 39 Sun X , Wang W , Dai J , et al. A long-term and slow-releasing hydrogen sulfide donor protects against myocardial ischemia/reperfusion injury . Sci Rep 2017 ; 7 : 3541 Google Scholar Crossref Search ADS PubMed WorldCat 40 Al-Magableh MR , Kemp-Harper BK , Hart JL. Hydrogen sulfide treatment reduces blood pressure and oxidative stress in angiotensin II-induced hypertensive mice . Hypertens Res 2015 ; 38 : 13 – 20 Google Scholar Crossref Search ADS PubMed WorldCat 41 Elsheikh W , Blackler RW , Flannigan KL , et al. Enhanced chemopreventive effects of a hydrogen sulfide-releasing anti-inflammatory drug (ATB-346) in experimental colorectal cancer . Nitric Oxide 2014 ; 41 : 131 – 7 Google Scholar Crossref Search ADS PubMed WorldCat 42 Sieghart D , Liszt M , Wanivenhaus A , et al. Hydrogen sulphide decreases IL-1beta-induced activation of fibroblast-like synoviocytes from patients with osteoarthritis . J Cell Mol Med 2015 ; 19 : 187 – 97 Google Scholar Crossref Search ADS PubMed WorldCat 43 Wallace JL , Vong L , McKnight W , et al. Endogenous and exogenous hydrogen sulfide promotes resolution of colitis in rats . Gastroenterology 2009 ; 137 : 569 – 78 Google Scholar Crossref Search ADS PubMed WorldCat 44 Xie L , Gu Y , Wen M , et al. Hydrogen sulfide induces Keap1 S-sulfhydration and suppresses diabetes-accelerated atherosclerosis via Nrf2 activation . Diabetes 2016 ; 65 : 3171 – 84 Google Scholar Crossref Search ADS PubMed WorldCat 45 Zhou X , An G , Lu X. Hydrogen sulfide attenuates the development of diabetic cardiomyopathy . Clin Sci (Lond) 2015 ; 128 : 325 – 35 Google Scholar Crossref Search ADS PubMed WorldCat 46 Yang G , Ju Y , Fu M , et al. Cystathionine gamma-lyase/hydrogen sulfide system is essential for adipogenesis and fat mass accumulation in mice . Biochim Biophys Acta 2018 ; 1863 : 165 – 76 Google Scholar Crossref Search ADS WorldCat 47 Tsai CY , Peh MT , Feng W , et al. Hydrogen sulfide promotes adipogenesis in 3T3L1 cells . PLoS One 2015 ; 10 : e0119511 Google Scholar Crossref Search ADS PubMed WorldCat 48 Cai J , Shi X , Wang H , et al. Cystathionine gamma lyase-hydrogen sulfide increases peroxisome proliferator-activated receptor gamma activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes . Biochim Biophys Acta 2016 ; 1861 : 419 – 29 Google Scholar Crossref Search ADS PubMed WorldCat 49 Geng B , Cai B , Liao F , et al. Increase or decrease hydrogen sulfide exert opposite lipolysis, but reduce global insulin resistance in high fatty diet induced obese mice . PLoS One 2013 ; 8 : e73892 Google Scholar Crossref Search ADS PubMed WorldCat 50 Wei WB , Hu X , Zhuang XD , et al. GYY4137, a novel hydrogen sulfide-releasing molecule, likely protects against high glucose-induced cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells . Mol Cell Biochem 2014 ; 389 : 249 – 56 Google Scholar Crossref Search ADS PubMed WorldCat 51 Szabo C. Roles of hydrogen sulfide in the pathogenesis of diabetes mellitus and its complications . Antioxid Redox Signal 2012 ; 17 : 68 – 80 Google Scholar Crossref Search ADS PubMed WorldCat 52 Morrow TJ. Animal models of painful diabetic neuropathy: The STZ rat model . Curr Protoc Neurosci 2004 ; Chapter 9, Unit 9 ; 1 8 . Google Scholar OpenURL Placeholder Text WorldCat 53 Muthuraman A , Diwan V , Jaggi AS , et al. Ameliorative effects of Ocimum sanctum in sciatic nerve transection-induced neuropathy in rats . J Ethnopharmacol 2008 ; 120 : 56 – 62 Google Scholar Crossref Search ADS PubMed WorldCat 54 Raz I , Hasdai D , Seltzer Z , et al. Effect of hyperglycemia on pain perception and on efficacy of morphine analgesia in rats . Diabetes 1988 ; 37 : 1253 – 9 Google Scholar Crossref Search ADS PubMed WorldCat 55 Tembhurne SV , Sakarkar DM. Effect of fluoxetine on an experimental model of diabetes-induced neuropathic pain perception in the rat Indian . Indian J Pharm Sci 2011 ; 73 : 621 – 5 Google Scholar Crossref Search ADS PubMed WorldCat 56 D’Mello R , Dickenson AH. Spinal cord mechanisms of pain BJA . Br J Anaesth 2008 ; 101 : 8 – 16 Google Scholar Crossref Search ADS PubMed WorldCat 57 Tölle TR , Berthele A , Zieglgänsberger W , et al. Flip and Flop variants of AMPA receptors in the rat lumbar spinal cord . Eur J Neurosci 1995 ; 7 : 1414 – 9 Google Scholar Crossref Search ADS PubMed WorldCat 58 Saadé NE , Jabbur SJ. Nociceptive behavior in animal models for peripheral neuropathy: Spinal and supraspinal mechanisms . Prog in Neurobiol 2008 ; 86 : 22 – 47 Google Scholar Crossref Search ADS WorldCat 59 Deleo JA , Colburn RW , Nichols ML , et al. Interleukin-6-mediated hyperalgesia/allodynia and increased spinal IL-6 expression in a rat mononeuropathy model . J Interferon Cytokine Res 1996 ; 16 : 695 – 700 Google Scholar Crossref Search ADS PubMed WorldCat 60 Takeda K , Sawamura S , Tamai H , et al. Role for cyclooxygenase 2 in the development and maintenance of neuropathic pain and spinal glial activation . Anesthesiol 2005 ; 103 : 837 – 44 . Google Scholar Crossref Search ADS WorldCat 61 Piao ZG , Cho IH , Park CK , et al. Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury . Pain 2006 ; 121 : 219 – 32 Google Scholar Crossref Search ADS PubMed WorldCat 62 Mika J , Osikowicz M , Makuch W , et al. Minocycline and pentoxifylline attenuate allodynia and hyperalgesia and potentiate the effects of morphine in rat and mouse models of neuropathic pain . Eur J Pharmacol 2007 ; 560 : 142 – 9 Google Scholar Crossref Search ADS PubMed WorldCat 63 Zurek JR , Nadeson R , Goodchild CS. Spinal and supraspinal components of opioid antinociception in streptozotocin-induced diabetic neuropathy in rats . Pain 2001 ; 90 : 57 – 63 Google Scholar Crossref Search ADS PubMed WorldCat 64 El-Khoury C , Hawwa N , Baliki M , et al. Attenuation of neuropathic pain by segmental and supraspinal activation of the dorsal column system in awake rats . Neuroscience 2002 ; 112 : 541 – 53 Google Scholar Crossref Search ADS PubMed WorldCat 65 Totsch SK , Sorge RE. Immune system involvement in specific pain conditions . Mol Pain 2017 ; 13 : 174480691772455 Google Scholar Crossref Search ADS WorldCat 66 Brownlee M. The pathobiology of diabetic complications: A unifying mechanism . Diabetes 2005 ; 54 : 1615 – 25 Google Scholar Crossref Search ADS PubMed WorldCat 67 McVicar CM , Hamilton R , Colhoun LM , et al. Intervention with an erythropoietin-derived peptide protects against neuroglial and vascular degeneration during diabetic retinopathy . Diabetes 2011 ; 60 : 2995 – 3005 Google Scholar Crossref Search ADS PubMed WorldCat 68 Zeng HY , Green WR , Tso MO. Microglial activation in human diabetic retinopathy . Arch Ophthalmol 2008 ; 126 : 227 – 32 Google Scholar Crossref Search ADS PubMed WorldCat 69 Avignone E , Lepleux M , Angibaud J , et al. Altered morphological dynamics of activated microglia after induction of status epilepticus . J Neuroinflam 2015 ; 12 : 202 . Google Scholar Crossref Search ADS WorldCat 70 Kim YS , Joh TH. Microglia, major player in the brain inflammation: Their roles in the pathogenesis of Parkinson's disease . Exp Mol Med 2006 ; 38 : 333 – 47 Google Scholar Crossref Search ADS PubMed WorldCat 71 Zhao QY , Xie XF , Fan YH , et al. Phenotypic dysregulation of microglial activation in young offspring rats with maternal sleep deprivation-induced cognitive impairment . Sci Rep 2015 ; 5 : 9513 Google Scholar Crossref Search ADS PubMed WorldCat 72 Chan WY , Kohsaka S , Rezaie P. The origin and cell lineage of microglia: New concepts . Brain Res Rev 2007 ; 53 : 344 – 54 Google Scholar Crossref Search ADS PubMed WorldCat 73 Whiteman M , Winyard PG. Hydrogen sulfide and inflammation: The good, the bad, the ugly and the promising . Expert Rev Clin Pharmacol 2011 ; 4 : 13 – 32 Google Scholar Crossref Search ADS PubMed WorldCat 74 Sodha NR , Clements RT , Feng J , et al. The effects of therapeutic sulfide on myocardial apoptosis in response to ischemia-reperfusion injury . Eur J Cardiothorac Surg 2008 ; 33 : 906 – 13 Google Scholar Crossref Search ADS PubMed WorldCat 75 Gui WS , Wei X , Mai CL , et al. Interleukin-1beta overproduction is a common cause for neuropathic pain, memory deficit, and depression following peripheral nerve injury in rodents . Mol Pain 2016 ; 12 : 174480691664678 Google Scholar Crossref Search ADS WorldCat 76 Eskan MA , Benakanakere MR , Rose BG , et al. Interleukin-1beta modulates proinflammatory cytokine production in human epithelial cells . Infect Immun 2008 ; 76 : 2080 – 9 Google Scholar Crossref Search ADS PubMed WorldCat 77 Rios EC , Soriano FG , Olah G , et al. Hydrogen sulfide modulates chromatin remodeling and inflammatory mediator production in response to endotoxin but does not play a role in the development of endotoxin tolerance . J Inflamm (Lond) 2016 ; 13 : 10 Google Scholar Crossref Search ADS PubMed WorldCat 78 Zhou YQ , Liu Z , Liu ZH , et al. Interleukin-6: An emerging regulator of pathological pain . J Neuroinflam 2016 ; 13 : 141 . Google Scholar Crossref Search ADS WorldCat 79 Schoeniger-Skinner DK , Ledeboer A , Frank MG , et al. Interleukin-6 mediates low-threshold mechanical allodynia induced by intrathecal HIV-1 envelope glycoprotein gp120 . Brain Behav Immun 2007 ; 21 : 660 – 7 Google Scholar Crossref Search ADS PubMed WorldCat 80 Fu Y , Xin Z , Liu B , et al. Platycodin D inhibits inflammatory response in LPS-stimulated primary rat microglia cells through activating LXRα-ABCA1 signaling pathway . Front Immunol 2017 ; 8 : 929 . Google Scholar Crossref Search ADS PubMed WorldCat 81 Renno WM , Alkhalaf M , Afsari Z , et al. Consumption of green tea alters glial fibrillary acidic protein immunoreactivity in the spinal cord astrocytes of STZ-diabetic rats . Nutr Neurosci 2008 ; 11 : 32 – 40 Google Scholar Crossref Search ADS PubMed WorldCat 82 Coleman E , Judd R , Hoe L , et al. Effects of diabetes mellitus on astrocyte GFAP and glutamate transporters in the CNS . Glia 2004 ; 48 : 166 – 78 Google Scholar Crossref Search ADS PubMed WorldCat 83 Rungger-Brandle E , Dosso AA , Leuenberger PM. Glial reactivity, an early feature of diabetic retinopathy . Invest Ophthalmol Vis Sci 2000 ; 41 : 1971 – 80 [Database] Google Scholar PubMed OpenURL Placeholder Text WorldCat 84 Asnaghi V , Gerhardinger C , Hoehn T , et al. A role for the polyol pathway in the early neuroretinal apoptosis and glial changes induced by diabetes in the rat . Diabetes 2003 ; 52 : 506 – 11 Google Scholar Crossref Search ADS PubMed WorldCat 85 Goodison KL , Parhad IM , White CL 3rd , et al. Neuronal and glial gene expression in neocortex of Down’s syndrome and Alzheimer’s disease . J Neuropathol Exp Neurol 1993 ; 52 : 192 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 86 Dennis JC , Coleman ES , Swyers SE , et al. Changes in mitotic rate and GFAP expression in the primary olfactory axis of streptozotocin-induced diabetic rats . J Neurocytol 2005 ; 34 : 3 – 10 Google Scholar Crossref Search ADS PubMed WorldCat 87 Mankovsky BN , Metzger BE , Molitch ME , et al. Cerebrovascular disorders in patients with diabetes mellitus . J Diabetes Complications 1996 ; 10 : 228 – 42 Google Scholar Crossref Search ADS PubMed WorldCat 88 Horani MH , Mooradian AD. Effect of diabetes on the blood-brain barrier . Curr Pharm Des 2003 ; 9 : 833 – 40 Google Scholar Crossref Search ADS PubMed WorldCat 89 McCall MA , Gregg RG , Behringer RR , et al. Targeted deletion in astrocyte intermediate filament (GFAP) alters neuronal physiology . Proc Natl Acad Sci USA 1996 ; 93 : 6361 – 6 . Google Scholar Crossref Search ADS PubMed WorldCat 90 Kakinuma Y , Hama H , Sugiyama F , et al. Impaired blood-brain barrier function in angiotensinogen-deficient mice . Nat Med 1998 ; 4 : 1078 – 80 Google Scholar Crossref Search ADS PubMed WorldCat 91 Abbott NJ. Astrocyte-endothelial interactions and blood-brain barrier permeability . J Anatomy 2002 ; 200 : 629 – 38 Google Scholar Crossref Search ADS WorldCat 92 Barber AJ , Antonetti DA , Gardner TW. Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes . Invest Ophthalmol Vis Sci 2000 ; 41 : 3561 – 8 Google Scholar PubMed OpenURL Placeholder Text WorldCat 93 Toran-Allerand CD , Bentham W , Miranda RC , et al. Insulin influences astroglial morphology and glial fibrillary acidic protein (GFAP) expression in organotypic cultures . Brain Res 1991 ; 558 : 296 – 304 Google Scholar Crossref Search ADS PubMed WorldCat 94 Reagan LP , Magarinos AM , Yee DK , et al. Oxidative stress and HNE conjugation of GLUT3 are increased in the hippocampus of diabetic rats subjected to stress . Brain Res 2000 ; 862 : 292 – 300 Google Scholar Crossref Search ADS PubMed WorldCat 95 Sandireddy R , Yerra VG , Areti A , et al. Neuroinflammation and oxidative stress in diabetic neuropathy: Futuristic strategies based on these targets . Int J Endocrinol 2014 ; 2014 : 1 – 10 Google Scholar Crossref Search ADS WorldCat 96 Rahman MH , Jha MK , Suk K. Evolving insights into the pathophysiology of diabetic neuropathy: Implications of malfunctioning glia and discovery of novel therapeutic targets . Curr Pharm Des 2016 ; 22 : 738 – 57 Google Scholar Crossref Search ADS PubMed WorldCat 97 Oguntibeju OO. Type 2 diabetes mellitus, oxidative stress and inflammation: Examining the links . Int J Physiol Pathophysiol Pharmacol 2019 ; 11 : 45 – 63 Google Scholar PubMed OpenURL Placeholder Text WorldCat 98 Volpe CMO , Villar-Delfino PH , Dos Anjos PMF , et al. Cellular death, reactive oxygen species (ROS) and diabetic complications . Cell Death Dis 2018 ; 9 : 119 Google Scholar Crossref Search ADS PubMed WorldCat 99 Khalfaoui T , Basora N , Ouertani-Meddeb A. Apoptotic factors (Bcl-2 and Bax) and diabetic retinopathy in type 2 diabetes . J Mol Hist 2010 ; 41 : 143 – 52 Google Scholar Crossref Search ADS WorldCat © 2020 American Association of Neuropathologists, Inc. All rights reserved. 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TI - Chronic Treatment With Hydrogen Sulfide Donor GYY4137 Mitigates Microglial and Astrocyte Activation in the Spinal Cord of Streptozotocin-Induced Diabetic Rats
JF - Journal of Neuropathology & Experimental Neurology
DO - 10.1093/jnen/nlaa127
DA - 2020-12-04
UR - https://www.deepdyve.com/lp/oxford-university-press/chronic-treatment-with-hydrogen-sulfide-donor-gyy4137-mitigates-PIagvG5dez
SP - 1320
EP - 1343
VL - 79
IS - 12
DP - DeepDyve
ER -