Low-dose chloroquine mediated neuroprotection against n-methyl-d-aspartate induced excitotoxicity in adult mice

Objective To investigate the neuroprotective effects of somatostatin and its effects on the expression of glial fibrillary acidic protein (GFAP) and synaptophysin(SYN) in rats with diabetic retinopathy. Methods A total of 24 clean male Sprague-Dawley(SD) rats were divided into control group, diabetic group and somatostatin treatment group according to random number table, with 8 rats in each group. Diabetes was induced by streptozotocin in diabetic group and somatostatin treatment group. After diabetes was induced, rats in somatostatin treatment group were given somatostatin (10 μg·kg-1·d-1) by intraperitoneal injection. Rats in control group and diabetic group were given the equal dose of saline. After 12 weeks, the density of retinal ganglion cells(RGC) were detected by HE staining; the expression of retinal GFAP and SYN were detected by immunofluorescence and Western blotting. Results Compared with control group, the fluorescence intensity of GFAP was significantly increased while the RGC density and the fluorescence intensity of SYN were decreased significantly. The expression of GFAP protein were increased and the expression of SYN protein was decreased in diabetic group compared with control group (F=187.62, 226.12, 159.30, 259.09, 425.93, all P<0.01). Compared with diabetic group, the fluorescence intensity of GFAP was decreased significantly while the RGC density and the fluorescence intensity of SYN were increased significantly. The expression of GFAP was decreased and the expression of SYN was increased in somatostatin treatment group compared with diabetic group (F=187.62, 226.12, 159.30, 259.09, 425.93, all P<0.01). Conclusion Somatostatin may increase the expression of SYN and restore the density of RGC by inhibiting the activation of glial cells, which suggests that somatostatin may play a neuroprotective role in diabetic retinopathy. Key words: Somatostatin; Diabetic retinopathy; Neuroprotection; Glial fibrillary acidic protein; Synaptophysin

Objective To observe the influence of human umbilical cord mesenchymal stem cells(hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP). Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A), diabetes mellitus (DM) only group (group B), DM + balanced salt solution (BSS) group (group C), DM + hUCMSC group (group D), with 10 rats in each group. DM rats were induced by intraperitoneal injection of streptozotocin. Apoptosis of retinal cells was assayed by dUTP nick end labeling. Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats. Results Compared with group A, large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C, however the apoptotic cells in group D were significantly reduced than group B and C. The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A, throughout the inner plexiform layer(IPL) in group B and C, only distributed in RNFL and GCL in group D. It was obvious that the expression of GFAP in group B and C was higher than group A. Compared with group B and C, the expression of GFAP in group D was significantly reduced. The difference of GFAP expression among the 4 groups was significant(F=79.635, P<0.05). Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats. Key words: Diabetic retinopathy; Mesenchymal stem cells; Neurons; Apoptosis

Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplantation into vitreous cavity of diabetic rats on the retinal morphology, and the expression of glial fibrillary acidic protein (GFAP) and rhodopsin (RHO). Methods 78 male Sprague-Dawley rats were used. 70 rats were injected with streptozotocin by tail vein injection at a dose of 40 mg/kg to establish the diabetes mellitus model, and another 8 rats were injected with 0.1 mol/L pH 4.0 citric acid buffer at the same dose as the normal control group. After 6 weeks of modeling, 10 rats were taken as the control group of diabetic model. hUCMSC suspension was injected into the right eye vitreous cavity of the remaining 60 rats, and the same volume of Dulbecco′s modified Eagle/F12 medium was injected into the left vitreous cavity as control eyes. 1, 2 and 4 weeks after transplantation, follow-up experiments were performed. The experimental eyes were labeled as U1, U2, and U4 groups, while the control eyes were recorded as D1, D2, D4, and each group consisted of 20 eyes. After paraffin section and hematoxylin-eosin staining, the structure of the retina was observed by optical microscopy and the thickness of the outer nuclear layer and the inner nuclear layer (INL) were measured. The distribution and migration of hUCMSC in rat retina were observed by frozen section-tissue immunofluorescence assay. The mRNA and protein expression of GFAP and RHO in the retina were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot assays. Results The results of optical microscope observation showed the normal structure of retina in normal control group. The retinal nerve fiber layer (NFL) was thinned and the number of retinal ganglion cells (RGC) in the control group of diabetic rats was decreased. The decreased number and disorder arrangement of RGC were observed as well in U1, D1 rats. The RGC number of U2, U4, D2, D4 rats was gradually decreased. Compared with D4 group, the thickness of INL in U4 group was significantly increased (P<0.05). Tissue immunofluorescence assay showed that hUCMSC were distributed along the inner limiting membrane in the retina of the U1 group, while the number of hUCMSC in the U2 group was gradually decreased, mainly in the NFL and ganglion cell layers. Real-time PCR and Western blot data indicated that the relative expression of GFAP mRNA and protein in the diabetic retina was significantly increased, and the relative expression of RHO mRNA and protein decreased gradually in the diabetic model group and the D1, D2, D4 groups. Compared with D2 and D4 groups, the mRNA and protein expression of GFAP in U2 and U4 groups were decreased, and the relative expression of RHO mRNA and protein were all increased (P<0.01). Conclusion hUCMSC could migrate and integrate into the retina, after the transplantation into the vitreous cavity of diabetic rats, which reduced the expression of GFAP, but enhanced the expression of RHO. Key words: Diabetic retinopathy/therapy; Mesenchymal stem cell transplantation; Rhodopsin; Animal experimentation

The relationship between glial fibrillary acidic protein (GFAP) expression and glial tumor cell behavior has not been well defined. The goal of this study was to examine this relationship further. To investigate the relationship between GFAP expression and glial tumor cell behavior, the authors isolated clones from the human glioblastoma cell line, U-373MG, according to their level of GFAP expression. Immunochemical analysis demonstrated that one clone had consistently low GFAP expression (approximately 93% of cells were GFAP negative), whereas a second clone had consistently high GFAP expression (approximately 80% of the cells were GFAP positive). The structure, population doubling time, saturation density, anchorage-independent growth, migratory rate, and invasive potential of these two clones were determined in relation to their level of GFAP expression. Morphologically, both clones were composed of ameboid as well as stellate components. Although the population doubling times of the two clones were equally rapid, the clone with low GFAP expression demonstrated a slightly higher saturation density compared with the clone with high GFAP expression. In an anchorage-independent environment (soft agar), a greater difference in growth characteristics was noted between the two clones: the high-expression clone formed more colonies and these colonies were compact, well defined, and spherical, whereas the low-expression clone formed predominantly smaller, two-dimensional colonies with vague boundaries and isolated cells or groups of cells at the periphery. In contrast to these minor differences between the clones, the low-expression clone showed a markedly increased migratory rate and invasive potential compared with the high-expression clone. Therefore, the clone with reduced GFAP expression appeared more aggressive, demonstrating decreased contact inhibition, increased migratory rate, and increased invasive potential. These results suggest a direct correlation between GFAP expression and some measures of aggressive tumor growth and transformation properties.

To explore the effect and potential mechanism of glycyrrhizin (GL) by inhibiting high mobility group box 1 (HMGB1) on glial scar formation after spinal cord injury (SCI) in rats. Seventy-two female Sprague Dawley rats were randomly divided into sham group ( n=12), SCI model group (SCI group, n=36), GL intervention group (SCI+GL group, n=12), and nuclear factor κB (NF-κB) inhibitor [pynolidine dithiocarbamate (PDTC)] intervention group (SCI+PDTC group, n=12). The SCI models of SCI group, SCI+GL group, and SCI+PDTC group were made by modified Allen's method, the sham group was only exposed the spinal cord without any injury. First of all, Basso-Beattie-Bresnahan (BBB) score of hind limbs and slope test were performed in SCI group at 1, 2, and 3 weeks after operation; Western blot was used to detect the expressions of glial fibrillary acidic protein (GFAP) and HMGB1 proteins. Compared with the sham group, the most significant time point in the SCI group was selected for subsequent experiment, in which the most significant glial scar was formed. Then, behavioral tests (BBB score of hind limbs and slope test), histological observation of spinal cord tissue structure, Western blot detection of HMGB1, GFAP, and NF-κB proteins, and immunohistochemical staining observation of GFAP and chondroitin sulfate proteoglycan (CSPG) were used to explore the effect of GL on the formation of glial scar after SCI and its potential mechanism. The BBB score and slope angle of the SCI group increased gradually with time, which were significantly lower than those of the sham group at each time point ( P<0.05). Western blot detection showed that the relative expressions of HMGB1 and GFAP proteins in the SCI group at 1, 2, and 3 weeks after operation were significantly higher than those in sham group ( P<0.05). The change was most obvious at 3 weeks after SCI, therefore the spinal cord tissue was selected for subsequent experiments at this time point. At 3 weeks after operation, compared with the SCI group, BBB score and slope angle of SCI+GL group significantly increased ( P<0.05); the relative expressions of HMGB1, GFAP, and NF-κB proteins detected by Western blot and the expressions of GFAP and CSPG proteins detected by immunohistochemical staining significantly decreased ( P<0.05); the disorder of spinal cord tissue by HE staining improved, inflammatory cell infiltration reduced, and glial scar formation decreased. At 3 weeks after operation, the expressions of NF-κB, GFAP, and CSPG proteins of the SCI+PDTC group significantly reduced when compared with the SCI group ( P<0.05); and the expression of NF-κB protein significantly decreased and the expressions of GFAP and CSPG proteins significantly increased when compared with the SCI+GL group ( P<0.05). After SCI in rats, the application of GL to inhibit the expression of HMGB1 can reduce the expression of GFAP and CSPG in the injured spinal cord, then reduce the formation of glial scars and promote the recovery of motor function of the hind limbs, and GL may play a role in inhibiting glial scar through HMGB1/NF-κB pathway.

Protective effects of Chinese herbal medicine Zuogui Pill on retina ganglion cells after optical nerve clipping injury in rats

Objective To observe the effect of netrin-1 on retinal Muller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Muller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=−26.01, 22.26; P=0.00, 0.00), and group E (t=−10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, −9.82; P=0.00, 0.00). There were no significant differences in between group A and group C (t=−0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Muller cells of DM rats could be decreased by injecting netrin-1 into vitreous. Key words: Diabetic retinopathy/physiopathology; Microglia/physiology; Nerve growth factors; Animal experimentation

Neuroprotective role of sphingolipid rheostat in excitotoxic retinal ganglion cell death

Objective: To investigate the effect of polydatin on retinal ganglion cell (RGC) apoptosis induced by optic nerve injury. Methods: It was an experimental research, conducted from October 2022 to December 2023. Retina-optic nerve explants from C57BL/6 mice were cultured in vitro to simulate optic nerve injury, and a computer-generated random number table was used for complete randomization, assigning the explants to the 0-day uncultured group (immediately detected after sampling), the model group (cultured for 1, 3 or 5 days to establish the injury model), and the polydatin group (with polydatin added throughout the intervention on the basis of the model group). The retinal tissues were collected, and the glial fibrillary acidic protein (GFAP), calcium ion binding protein 1 (Iba1), and ganglion cell marker Brn3a were detected by immunofluorescence staining. The effects of polydatin on the activation of astrocytes and microglia and RGC survival were observed. The expressions of GFAP, Vimentin, Iba1, B-lymphocytoma-2-associated X protein (Bax), lyzed C-caspase 3, and B-lymphocytoma-2 were detected by Western blotting. Real-time fluorescence quantitative PCR was used to detect the mRNA transcription levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10. One-way analysis of variance and LSD-t test were used for statistical analysis. Results: Immunofluorescence staining showed that compared with the model group, the polydatin group inhibited the activation of astrocytes and microglia and protected the RGCs. With explant culture for 1, 3, and 5 days, the expressions of GFAP (0.74±0.01, 0.70±0.04, 0.68±0.02), Vimentin (0.67±0.02, 1.91±0.09, 1.25±0.05), Iba1 (0.87±0.10, 2.36±0.13, 1.64±0.11), Bax (2.48±0.10, 0.37±0.02, 1.69±0.11), and C-caspase 3 (0.77±0.03, 2.49±0.09, 1.65±0.08) in the polydatin group were lower than those in the model group [GFAP (1.23±0.01, 1.17±0.01, 1.77±0.04), Vimentin (1.21±0.02, 2.67±0.06, 1.42±0.03), Iba1 (1.13±0.02, 3.51±0.07, 2.16±0.08), Bax (3.53±0.12, 1.27±0.06, 3.24±0.15), and C-caspase 3 (1.54±0.08, 3.38±0.17, 2.18±0.08)]. The expression of B-lymphocytoma-2 in the polydatin group (2.41±0.09, 1.67±0.07, 6.84±0.20) was higher than that in the model group (1.73±0.08, 0.96±0.07, 2.36±0.33). The differences were statistically significant (all P<0.05). In addition, the mRNA transcription levels of TNF-α (0.47±0.13, 12.07±0.56, 18.06±2.58) and IL-6 (0.55±0.12, 7.48±1.02, 41.35±7.08) in the polydatin group were lower than those in the model group [TNF-α (4.67±0.52, 26.62±2.62, 42.43±4.97) and IL-6 (1.21±0.06, 15.66±0.62, 67.46±3.78)]. The level of IL-10 (0.52±0.07, 2.98±0.24, 5.61±1.23) in the polydatin group was higher than that in the model group (0.06±0.03, 0.12±0.03, 2.64±0.74). The differences were statistically significant (all P<0.05). Conclusion: Polydatin can inhibit the activation of glial cells and the expression of inflammatory factors induced by optic nerve injury, thus enhancing the survival of RGCs.

Objectives: Canine distemper virus (CDV), a member of the genus Morbillivirus of the family Paramyxoviridae, is the causative agent of canine distemper, a fatal and highly contagious disease that affects dogs and other carnivores. This study aimed to investigate whether there is a correlation between glial fibrillary acidic protein (GFAP) and neurofilament (NF) expression in canine distemper encephalomyelitis (CDE) and the severe neuropathology that occurs. Materials and Methods: GFAP and NF expression levels in the brain tissue of 13 dogs diagnosed with CDE were investigated by immunohistochemical method. Results: The results of the study revealed that GFAP (P &lt; 0.005) and NF (P &lt; 0.005) expression levels in brain tissue were significantly increased in CDV-infected dogs compared to healthy, uninfected dogs. GFAP expression was mainly observed in endothelial cells and astrocytes, whereas NF expression was mainly found in neurons. In addition, it was found that the expression of both GFAP and NF was more pronounced in the areas with the most severe neuropathological findings. Conclusions: This study demonstrated pathological astrocyte reactivation and neuronal degeneration at the molecular level. These findings provide information about the stage of the disease. This study clearly demonstrated that detailed information about the prognosis of the disease can be obtained from GFAP and NF expression. Since GFAP/NF levels provide information about the severity of the disease, they can be used clinically. Therefore, further research into the involvement of GFAP and NF expression in the pathophysiology of CDE has great potential to improve our understanding of this complex neurological disorder.

Background/aim The effects of systemic magnesium sulfate (MgSO4) on retina in preterm hypoxic-ischemic (HI) rat model are not known. Our aim was to investigate the effects of MgSO4 on retinal ganglion cell (RGC) count, retinal ganglion cell (RGC) apoptotic index, retinal vascular endothelial growth factor receptor-2 (VEGFR-2), and glial fibrillary acidic protein (GFAP) expressions in preterm HI rat model.Materials and methods Fifteen, postnatal day (PND) 7 rat pups were divided into 3 groups: 1. Sham-operated group, 2. HI group, and 3. MgSO4-treated HI group. The second and third groups underwent ischemia followed by exposure to hypoxia for 2 h (Vannucci model). The first and second groups received intraperitoneal saline and the third group received intraperitoneal MgSO4. On PND 10, eyes of the pups were evaluated for RGC count, apoptotic index, VEGFR-2, and GFAP expressions. Results In both HI and MgSO4-treated HI group, the mean total RGC counts were found to be significantly decreased. However, the mean total RGC count in the MgSO4-treated HI group was significantly higher than that of the HI group. The mean apoptotic index was found to be significantly increased in the HI group. Retinal VEGFR-2 and GFAP expressions were found to be significantly higher in the HI group.ConclusionsMagnesium sulfate preconditioning andtreatment in preterm HI rat model might diminish apoptosis, relatively preserve RGCs, and reduce retinal VEGFR-2 and GFAP expressions.

Objective To evaluate the role of spinal nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in hydrogen-induced reduction of inflammatory pain in rats. Methods Sixty-four SPF healthy adult male Sprague-Dawley rats, weighing 200-250 g, were divided into 4 groups (n=16 each) using a random number table: control group (group C), inflammatory pain group (group IP), inflammatory pain plus hydrogen-rich saline group (group IP+ H2) and inflammatory pain plus hydrogen-rich saline plus Nrf2 inhibitor all-trans retinoic acid (ATRA) group (group IP+ H2+ ATRA). Chronic inflammatory pain was induced by injecting complete Freund′s adjuvant (CFA) 100 μl into the plantar surface of the left hind paw in IP group and IP+ H2 group.The equal volume of normal saline was given instead in group C. Hydrogen-rich saline 5 ml/kg was injected intraperitoneally once a day for 7 consecutive days starting from 1 day after injecting CFA in group IP+ H2 and group IP+ H2+ ATRA, and the equal volume of normal saline was given instead in the other groups.ATRA 7 mg/kg was injected intraperitoneally once a day for 2 consecutive days starting from 2 days before injecting CFA.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before establishing the model (T0) and 1, 3 and 7 days after establishing the model (T1-3). Six rats were sacrificed after the last measurement of pain threshold on day 7 after establishing the model, and the L4-6 lumbar segments of the spinal cord were removed for determination of the expression of Nrf2, HO-1 and glial fibrillary acidic protein (GFAP) by Western blot. Results Compared with group C, the MWT was significantly decreased and the TWL was shortened at T1-3, and the expression of Nrf2, HO-1 and GFAP was up-regulated in IP and IP+ H2 groups (P 0.05). Compared with group IP+ H2, the MWT was significantly decreased and the TWL was shortened at T1-3, the expression of Nrf2 and HO-1 was down-regulated, and the expression of GFAP was up-regulated in group IP+ H2+ ATRA (P<0.05). Conclusion Activation of spinal Nrf2/HO-1 signaling pathway is involved in hydrogen-induced reduction of inflammatory pain in rats. Key words: NF-E2-related factor 2; Heme oxygenase-1; Spinal cord; Hydrogen; Pain; Inflammation

Background Oxidative stress is one of the pathogenesis mechanisms of diabetic complications.Melatonin is the most powerful antioxidant in living organism.Seldom study on the effects of melatonin on diabetic complications is found in China now.Objective This study aimed to detect the expression and alteration of malonadehyde(MDA),glutathione(GSH)and glial fibrillary acidic protein(GFAP)in retina with experimental diabetes and explore the effects of melatonin on retinal oxidative stress in diabetic rats. Methods The animal models of diabetes were established by injection of 2％ streptozocin via caudal vein(STZ,45 mS/kg)in 48 6-week-old clean SD rats.Isometric citric acid buffer solution wag injected in 24 normal.rats as control group.Seven days after injection of STZ,melatonin(10 mg/kg)was injected into the abdominal cavity of 24 diabetic rats in melatonin group dailv,and the equal volume of normal saline was injected in other 24 diabetic rats(model rats)and 24 rats in normal control group.The animals were sacrificed and retinas were obtained in three groups in 4,6 and 8 weeks respectively.Immunohistochemistry and Western blot were used to detect the expression of GFAP in rat retina.The levels of MDA and GSH in retina homogenate were assayed by spectrophotometer.This experiment complied the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The expression of GSH in model group was lower than that in melatonin group or control group at 4,6 and 8 weeks (all P 0.05),and in the eighth week,expression of GSH in melatonin group was lower than that in control group(P 0.05).The expressions of MDA and GFAP in melatonin group were higher than those in control group at 8 weeks(P<0.05).Conclusion Melatonin plays a powerful effect in protecting the retina from oxidative stress in diabetes retina by downregulating the MDA level,upregulating GSH level and inhibiting GFAP expression. Key words: Melatonin; Diabetic retinopathy; Malonaldehyde; Glutathione; Glial fibrillary acidic protein

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

Maternal food deprivation in F0 generation and hypercaloric diet in F1 generation reduced GFAP expression after an immune challenge in F2 generation in several brain areas