Altered expression of G protein-coupled inwardly rectifier potassium channels (GIRK) subunit 1 and 2 in hippocampus of chronic temporal epileptic rats induced by kainic acid

AIM: G protein-coupled inwardly rectifier potassium (GIRK) channel are distributed widely in mammalian brain. In CNS, GIRK 1/2 seems to be the predominant heterotetramers which play a pivot role in the regulation of the excitability of neurons and may contribute to the resting potential by leading to a hyperpolarization of membrane potential and reduction of the action potential frequency. In the context, the Weaver mouse is the first neurological abnormality directly linked to a genetic point mutation in the GIRK2 protein which includes spontaneous seizure. GIRK2 knock out mice showed normal development but more susceptible than normal mice to seizure induced by GABA antagonist. Here, we report that the mRNA and protein expression of GIRK subunit 2 is altered in kainic acid(KA)-induced epileptic rat hippocampus. METHODS: Rats were injected with kainate 14 mg/kg intraperitoneally to establish an acute and chronic temporal lobe epilepsy model. At chronic spontaneous seizure stage, by using of in situ hybridization, immunocytochemistry and Western blotting, the GIRK 1,2 mRNA and protein were analyzed quantitatively in the dentate gyreus, CA1, CA3 regions of hippocampus. RESULTS: GIRK1,2 mRNA and proteins were expressed abundantly in all regions of hippocampus. KA induced seizures and caused a significant increase in GIRK2 mRNA abundance and immunoreacitivity; only GIRK1 mRNA was increased significantly, but no difference was found by Western blotting protocol. CONCLUSION: GIRK1,2 mRNA and protein expression in the hippocampus of epileptic rat brain is up-regulated, which may be an adaptive response to over-excitability of neuron networks and prevent the over-excitability spread in hippocampus (DG-CA3-CA1).     

Effects of SAHA on hippocampal TLR4/MYD88 signaling and neuronal apoptosis after seizure in developing rats

AIM: To investigate the effect of vorinostat(suberoylanilide hydroxamic acid, SAHA), a histone deacetylase inhibitor, on seizure-induced brain damage in developing rats and its mechanism. METHODS: Male Sprague-Dawley rats(n=32) were randomly divided into control group, pentylenetetrazole(PTZ) group, PTZ+10 mg/kg SAHA group and PTZ+50 mg/kg SAHA group. Intraperitoneal injection of PTZ was used to induce rat seizure. SAHA was injected intraperitoneally 2 h before PTZ injection. The rats in different seizure stages were counted and mean seizure score was analyzed at 30~60 min after PTZ injection. Hippocampal tissues were sampled at 24 h after seizures. The expression of TLR4, MYD88, NF-κB P65 and IL-1β at mRNA and protein levels was detected by RT-qPCR and Western blot, respectively. The pathological changes of the brain tissues were observed by HE staining. The apoptotic neurons were observed by TUNEL staining. RESULTS: The mRNA and protein levels of TLR4, MYD88, NF-κB P65 and IL-1β, the apoptosis of neurons, the inflammation reaction and mean seizure score significantly increased after PTZ treatment(P<0.05), and these effects were attenuated by treatment with SAHA. Compared with PTZ+10 mg/kg SAHA group, PTZ+50 mg/kg SAHA group showed more significant protective effect against seizure-induced brain damage. CONCLUSION: Histone deacetylase inhibitor SAHA suppresses seizure-induced TLR4/MYD88 signaling and reduces apoptosis of neurons, suggesting a protective effect against brain damage associated with seizure in developing rats.     

Expressions of EphA4 during apoptosis in rat cerebral cortical neurons induced by kainic acid and effect of gastrodin

AIM: To explore the expressions of EphA4 during apoptosis in rat cerebral cortical neurons induced by kainic acid(KA) and the effect of gastrodin on expressions of EphA4. METHODS: Neurons from newborn rat cerebral cortex were cultured in vitro for seven days, and were divided into three groups at random: control group, exposure to KA group, and exposure to KA and gastrodin group. Morphologic changes of neurons were observed with inverted phase contrast microscope. Apoptosis and necrosis of neurons were detected by staining of fluorescein Hoechst 33258 or AO/EB, and measurement of LDH. Expressions of EphA4 were measured by specific antibody labeled with CY3. RESULTS: Neuronal apoptotic ratios were notably increased and expressions of EphA4 were notably up-regulated in exposure to KA group compared to control group. Neuronal apoptotic ratios were notably decreased and up-regulations of EphA4 were notably inhibited in exposure to KA and gastrodin group compared to exposure to KA group. CONCLUSION: The expression of EphA4 is increased during apoptosis in rat cerebral cortex neurons induced by KA. Gastrodin inhibits the expression of EphA4 and has a neuroprotective effect during this process.     

Effect of electroacupuncture at auricular concha on behaviors and electroencephalogram in epileptic rats

AIM: To explore the antiseizure mechanism of electroacupuncture at auricular concha (AC).METHODS: Forty-eight healthy adult male rats were divided into 2 parts for behavioral observation (n=24) and electrophysiological observation (n=24), respectively. For behavioral observation, 24 rats were divided into 3 groups: model group (n=8), Dazhui group (Du 14, n=8) and AC group (n=8). The rats in model group were treated with intraperitoneal injection of pentylenetetrazol (PTZ, 60 mg/kg). The rats in the latter 2 groups were pretreated with electroacupuncture at "Du 14" and AC, respectively, and then received intraperitoneal injection of PTZ. Behavioral observations were performed to determine the antiseizure effect of electroacupuncture at AC. For electrophysiological observation, 24 rats were divided into 3 groups: Dazhui group (n=8),vagus nerve stimulation (VNS) group (n=8) and AC group (n=8). The rats in Dazhui group were treated with electroacupuncture at "Du 14". The rats in VNS group were given cervical vagus nerve stimulation. The rats in AC group were treated with electroacupuncture at AC. Epileptic discharges in electroencephalogram were observed to determine the effect of electroacupuncture at AC for the treatment of epilepsy. RESULTS: Compared with model group, the latency of the first grand mal seizure increased in AC group (P<0.05 or P<0.01). Compared with Dazhui group, the latency of the first grand mal seizure increased in AC group (P<0.05). Compared with model group, the durations of the first grand mal decreased in Dazhui group and AC group (P<0.05 and P<0.01, respectively). Compared with model group, the scores of epileptic behaviors decreased in AC group (P<0.01). Compared with Dazhui group, the scores of epileptic behaviors decreased in AC group (P<0.01). The antiseizure duration by VNS and electroacupuncture at AC increased, compared with that by electroacupuncture at "Du 14" (P<0.01). No statistical difference between the antiseizure duration of VNS and electroacupuncture at AC was observed (P>0.05). CONCLUSION: The antiseizure effect of electroacupuncture at AC is better than that of electroacupuncture at "Du 14". There is no significant difference between the acute antiseizure duration of VNS and electroacupuncture at AC. Electroacupuncture at AC is effective, convenient and low-cost, which may be used as a complementary therapy for epilepsy.     

Effects of synthetic kainic acid on microfilament expression and gap junction in astrocytes

AIM：To study the effects of synthetic kainic acid (SKA) and 1-heptanol (1-Hep), a gap junction blocker, on the cytoskeletal filament expression in the astrocytes. METHODS：The neonatal rat brain was obtained from the Wistar rats (1 day old) and primary purified astrocytes were obtained by differential attachment for removing filamentoblasts and orbital shaker for removing the oligodendrocytes. The effects of SKA and other interventions on the morphologic changes and expression levels of skeleton protein filamentous actin (F-actin) were observed in the astrocytes after 24 h of the exposures by laser scanning confocal microscopy. The effect of 1-Hep on the expression of F-actin was also explored. RESULTS：Compared with control group, the fluorescence intensities in KCl group and KCl+SKA group were increased, and highly increased in KCl+SKA group. The F-actin filaments in the above 2 groups were more intensive, thickened and concentrated than those in control group, and more obvious in KCl+SKA group. l-Hep significantly decreased the expression of F-actin in KCl group and KCl+SKA group as compared with control group, and parts of the filamentous fracture were seen in the astrocytes in all 1-Hep-treated groups, in which some of the filamentous lines were crosscut. CONCLUSION：Increase in the expression of F-actin in the astrocytes affects the structure and function of the intercellular gap junctions, which may be involved in the mechanism of SKA-induced epilepsy.     

Effects of piceatannol on rat kidney with diabetic nephropathy in early stage

AIM: To observe the effect of piceatannol on the kidney of diabetic nephropathy rats in early stage, and to explore the possible mechanisms.METHODS: The rats were randomly divided into 5 groups:control group, model group, low dose of piceatannol treatment group, medium dose of piceatannol treatment group and high dose of piceatannol treatment group. The rat model of diabetic nephropathy was induced accordingly, and the rats received 20 mg/kg, 40 mg/kg or 60 mg/kg of piceatannol by gavage once a day for 4 weeks. Blood glucose was detected by glucometer. The urea nitrogen and creatinine levels in the serum were measured by urease-glutamate dehydrogenase enzymatic and inosine acid oxidase methods, respectively, and 24 h urinary microalbumin was analyzed by immune transmission turbidimetry test. Moreover, the pathological changes of the kidney tissues were observed under microscope with HE staining. The protein expression of TGF-β1 and Smad 7 and the phosphorylation levels of Smad2 and Smad3 were determined by Western blot. RESULTS: Compared with model group, piceatannol treatment significantly decreased the levels of blood glucose, blood urea nitrogen and urinary microalbumin, but had no effects on serum creatinine. Furthermore, HE staining showed that the increased mesangial cells, matrix hyperplasia and degenerated epithelial cells in model group were markedly inhibited after piceatannol treatment. Additionally, piceatannol treatment also reduced the protein expression of TGF-β1 and Smad 7, and the phosphorylation levels of Smad2 and Smad3. CONCLUSION: Piceatannol attenuates pathological progression in the kidney of diabetic nephropathy rats in early stage, which may be through inhibiting TGF-β/Smad signaling pathway.     

Ferulic acid protects against apoptosis of PC12 cells induced by kainic acid

AIM：To investigate the effect of ferulic acid (FA) on the apoptosis of PC12 cells induced by kainic acid (KA) in vitro. METHODS：In order to establish an Alzheimer disease neuronal cell model, the rat pheochromocytoma cell line PC12 was treated with KA at a concentration of 50 μmol/L. These model neurons were divided into KA model group and 3 groups treated with FA at doses of 25, 50 and 100 μmol/L, respectively. At the same time, normal group was established without KA pretreatment. The viability of the PC12 cells was detected by MTT assay. The expression of Bcl-2, Bax and cytochrome C (Cyt C) was determined by immunocytochemical method. Apoptotic rate of the PC12 cells was measured by flow cytometry with annexin V/PI double staining. The protein levels of Bcl-2, Bax and Cyt C were analyzed by Western blotting. RESULTS：The cell survival rate, the expression of Bcl-2 and the ratio of Bcl-2 to Bax in KA model group were significantly decreased (P<0.01),while the expression of Bax and Cyt C was obviously increased compared with normal control group (P<0.01). The apoptotic rate in KA model group was obviously increased compared with normal control group (P<0.01) After the intervention of FA, the cell survival rates were increased and the apoptotic rates were decreased. Furthermore, the positive rate and expression of Bcl-2, and the ratio of Bcl-2 to Bax in each dose of FA treatment group were significantly increased, while the expression of Bax and Cyt C in each dose group was significantly reduced as compared with KA model group (P<0.05 or P<0.01). CONCLUSION：KA obviously induces apoptosis of PC12 cells. FA had obvious protective effect on PC12 cells against the toxicity of KA. FA blocks endogenous apoptic pathway through inhibiting the expression of Bax and Cyt C and increasing the expression of Bcl-2 and the ratio of Bcl-2/Bax, thus improving the survival rate of PC12 cells.     

Effect of Ganoderma lucidum spores on cytochrome C,mitochondrial Ca2+, HSP70 and BDNF in brain of epilepsy rats

AIM: To investigate the effects of Ganoderma lucidum spores on superoxide dismutase(SOD),malondialdehyde(MDA),total antioxidative capacity(T-AOC), cytochrome C, heat-shock protein 70 (HSP70), mitochondrial Ca²⁺ and brain-derived neurotrophic factor(BDNF) in the brain tissues of epilepsy rats.METHODS: The rat chronic epilepsy model was by intraperitoneal injection of pentetrazole(PTZ) at a subconvulsant dose (32 mg/kg).Flame atomic absorption method was used to detect the content of mitochondrial Ca²⁺,and spectrophotometer colorimetry was used to measure SOD activity,MDA content,T-AOC and cytochrome C levels in rat brain tissues. HSP70 and BDNF were determined by immunohistochemical method.RESULTS: The contents of mitochondrial Ca²⁺ and cytochrome C were higher, and the content of intracytoplasmic cytochrome C in the rat brain tissues was obviously lower in Ganoderma lucidum spores group than that in epileptic model group. Compared to epileptic model group, the activity of SOD and T-AOC in cytoplasm of the rat brain tissues decreased while MDA increased, and the numbers of BDNF-positive cells in cerebral cortex and hippocampus were significantly increased in Ganoderma lucidum spores group. The positive neuron population of HSP70 in hippocampus, basal nucleus and cortex was significantly higher in Ganoderma lucidum spores group than that in epilepsy model group.CONCLUSION: Ganoderma lucidum spores attenuate the impairment of neuronal mitochondria induced by seizure, and accelerate the expression of BDNF, resulting in restoring the energy metabolism in mitochondrion, thus alleviating the impairment and apoptosis of the brain tissues.     

Neuroprotective effect of wortmannin on epileptic rats by inhibiting autophagy

AIM: To investigate the role of autophagy in hippocampus injury induced by seizures and to observe the neuroprotective effects of autophagy inhibitor wortmannin(WM) on epileptic rats.METHODS: The Wistar rats were randomly divided into control group, model groups at 2 h, 8 h, 16 h, 24 h or 72 h after seizure induction by pilocarpine, and WM pretreatment group. The methods of HE and Nissl staining were used to evaluate the hippocampus injury. The expression of microtubule-associated protein 1 light chain 3(LC3) was detected by Western blotting. The ratio of LC3II to LC3I was calculated and used to represent the activity of autophagy. RESULTS: The significant increase in the ratio of LC3II to LC3I began at 2 h, peaked at 24 h, and maintained at high level at least to 72 h after seizure induction. Obvious neural injury and neuron depletion were observed in hippocampus CA1 area at 24 h after seizure induction. The number of surviving neurons at 24 h was sharply decreased in rats with seizures(75.50±5.92) as compared to the controls(110.67±18.56, P＜0.01). WM significantly decreased the neuron depletion induced by seizures(100.88±18.73, P＜0.05). Moreover, WM significantly decreased the ratio of LC3II to LC3I in rats with seizures at 24 h(P＜0.05). CONCLUSION: Autophagy was activated in hippocampus injury induced by seizures. WM reduces the transformation of LC3II to LC3I to inhibit the autophagy activated by seizures. WM has neuroprotective effect on epileptic rats by increasing the surviving neurons in hippocampus CA1 area.     

Effect of Tripterygium hypoglaucum Hutch on MMP-13 protein expression and IL-12, IL-23 and IL-37 levels in serum and foot paws of rats with collagen-induced arthritis

AIM: To investigate the effect of tripterygium hypoglaucum Hutch (THH) on collagen-induced arthritis (CIA) in rats and its possible mechanism. METHODS: SD rats were randomly divided into normal group and CIA group. The rat model of type Ⅱ CIA was successfully established and the model rats were randomly divided into 4 different groups: model group, dexamethasone group, THH (200 mg/kg) group, and THH (400 mg/kg) group. The contents of IL-12, IL-23 and IL-37 in the serum and foot paws of the CIA rats were detected by ELISA. The histopathological changes of the skin of the food paws were observed by HE staining. The protein expression of MMP-13 was determined by Western blot. The MMP-13 activity in the foot paws was detected by fluorescence labeling method. RESULTS: Compared with CIA group, THH at dose of 400 mg/kg significantly reduced the weight loss in type Ⅱ CIA rats (P<0.01). THH at dose of 400 mg/kg obviously decreased the contents of IL-12 by 28.31%, IL-23 by 41.57% in the serum and IL-12 by 30.78%, IL-23 by 39.46% in the foot paws, while IL-37 was significantly increased by 79.43% in the serum and 75.78% in the foot paws (P<0.01). The pathological changes of the subcutaneous tissues were improved by treating with THH (400 mg/kg). The protein expression of MMP-13 was significantly decreased by 31.82% (P<0.01), and the MMP-13 activity was also reduced. THH at dose of 200 mg/kg had no obvious improvement on the above indexes. CONCLUSION: THH has significant inhibitory effect on rat CIA by reducing the content of proinflammatory cytokines IL-12 and IL-23, increasing the content of anti-inflammatory factor IL-37, inhibiting inflammatory cell infiltration and vascular proliferation, and attenuating the protein expression of MMP-13 and MMP-13 activity in rats.     

Expression of EAAT3 in prefrontal cortex and hippocampus in CPP reinstatement rats induced by morphine

AIM: To investigate the role of excitatory amino acid transporter 3(EAAT3) in prefrontal cortex and hippocampus in morphine relapse by detecting the changes of EAAT3 expression in prefrontal cortex and hippocampus in conditioned place preference (CPP) reinstatement rat model induced by morphine.METHODS: Forty adult male SD rats, weighing 200-250 g, were randomly divided into 5 groups with 8 rats each: control group, CPP establishment group (Es), CPP extinction group (Ex), reinstatement 2 h group (Re2) and reinstatement 4 h group (Re4).Intraperitoneal (ip) injection of morphine was applied at a constant dose (10 mg/kg) for 10 days to the established CPP model.Normal saline instead of morphine was used to induce CPP extinction for 10 days.CPP was reinstated following a single priming injection of morphine (2.5 mg/kg).After the CPP behavior test, the rats were sacrificed, and the prefrontal cortex and hippocampus were collected for detecting the levels of EAAT3 by Western blotting.RESULTS: The accumulated time the rats spent in the drug-paired chamber was significantly longer in Es group, Re2 group and Re4 group than that in control group (P<0.05).Compared with control group, the expression of EAAT3 in prefrontal cortex significantly decreased both in Es group and Re4 group (P<0.05).No significant change of EAAT3 in hippocampus among groups was observed (P>0.05), while EAAT3 in hippocampal CA1 area significantly increased in Es group and Ex group as compared with control group (P<0.05).CONCLUSION: The expression of EAAT3 in prefrontal cortex decreases both in CPP establishment and reinstatement models, indicating that down-regulation of EAAT3 in prefrontal cortex may partly participate in the formation of opium relapse.     

Improvement of vascular hyporesponsiveness in rats with sepsis by protein C activator from Agkistrodon acutusvenom

AIM:To investigate the effects of protein C activator (PCA) from Agkistrondon acutusvenom (AAV) on the tension of thoracic aorta rings isolated from the rats with sepsis. METHODS:The model of sepsis was established by intraperitoneal injection of lipopolysaccharide (LPS). SD rats were randomly divided to 6 groups (n=6): sham group, LPS group, PCA intervention group (LPS+PCA, PCA at doses of 0.1 mg/kg, 0.3 mg/kg and 0.6 mg/kg) and LPS+ polymyxin B (at dose of 0.2 mg/kg) group. Using perfusion experiment in vitro, the tension of the aortic rings was measured by biological signal analytical system. RESULTS:The values of MABP, HR, LVDP and ±dp/dtmax were significantly lower in LPS group than those in sham group and LPS+PCA groups. Compared with sham group, the relaxation response to acetylcholine (ACh) and the contractile response of aorta rings induced by phenylephrine (Phe) were significantly decreased in LPS group, which were increased significantly in PCA intervention group (especially at dose of 0.6 mg/kg) compared with LPS group. The dose-response curve of aorta contraction with denuded endothelium induced by Phe shifted down significantly in LPS group compared with sham group, and no significant difference between LPS group and PCA intervention group was observed. Also no statistical difference was found in non-endothelium dependent relaxation of aortic rings induced by sodium nitroprusside among the groups. Pretreatment of N-nitro-L-arginine methl ester and methylene blue increased the contraction amplitude of aortic rings induced by Phe. CONCLUSION: PCA from AAV effectively reverses the hypoergia of the vessels in rats with sepsis through protecting vascular endothelium, the mechanism of which may be mediated by inhibiting NO-GC-cGMP signal transduction pathway.     

Changes of IL-4, IL-10, IL-12 levels in rat serum and lung during acute respiratory distress syndrome induced by oleic acid

AIM:To observe the dynamic changes of interleukin-4(IL-4), IL-10, IL-12 in rat serum and lung tissues during acute respiratory distress syndrome (ARDS).METHODS:The ARDS model of rats was induced by intravenous injection of oleic acid. The levels of IL-4, IL-10, IL-12 in serum and the supernatant of lung tissues were measured by enzyme linked immunosorbent assay (ELISA).RESULTS:The Levels of serum and lung IL-10, IL-12 in ARDS rats were increased in 4 h, 8 h, 16 h group compared with control group. The levels in IL-10 in serum in 16 h group and IL-10 in lung tissues of 8 h group were lower than that in 4 h group. The Levels of IL-4 in serum in 4 h, 8 h group were higher than that in control group, while IL-4 in 16 h group was lower than that in 8 h group. IL-4 of lung tissues in 4 h, 8 h, 16 h group were increased significantly, but in 16 h group were lower than that in 8 h group. The biggest changes of pulmonary coefficient and histopathology were observed at 4 h after injection of oleic acid.CONCLUSIONS:IL-4, IL-10 and IL-12 might play important roles in inflammatory reaction induced by oleic acid. The pro-and anti-inflammatory cytokines produced successively during ARDS.The relationship between unbalanced cytokines and lung injury in ARDS needs to be further studied.     

PHC inhibits the defects of alveolar type II epithelial cells and activation of ERK in lung tissue of ALI rat induced by LPS

AIM: To investigate the effects of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS) induced the changes of ultrastructure of alveolar type II epithelial cells (ATII) and activation of extracellular signal-regulated protein kinase (ERK) in lung tissue in rats. METHODS: Acute lung injury (ALI) was induced successfully by intravenous administration of LPS (5 mg/kg) in rats. PHC (3.0, 1.0, and 0.3 mg/kg) was administered to rats 0.5 h prior and then again concomitant with LPS exposure. The changes of ultrastructure of ATII, lung permeability index (LPI), wet to dry weight (W/D) ratio in lung were measured at 6 h after LPS application. Western blotting analysis was performed to determine the phosphorylations of ERK in lung tissue at 6 h after LPS application. To examine whether the effects of PHC on activation of ERK was in a time-dependent manner, lung tissues at 0 h, 2 h, 4 h, 6 h, and 12 h were collected for measuring the level of phosphorylated ERK. RESULTS: Challenge with LPS alone resulted in a significant increase in W/D ratio in lung and LPI. The defects of ATII with no lamellar bodies in cytoplasm, the lack of microvilli along its margin, severely swollen endoplasmic reticulum, nuclear cisterna and loss of integrity of the basement membrane induced by LPS were observed under transmission electron microscope. LPS also triggered activation of ERK at 2 h. Pre-treatment with PHC significantly abolished increase in W/D ratio in lung, LPI and attenuated pathological changes of ATII in a dose-dependent manner. Moreover, pre-treatment with PHC efficiently blunted the activation of ERK induced by LPS at 6 h. CONCLUSION: These results suggest that pre-treatment with PHC significantly attenuates the lung permeability and defects of ATII in LPS-induced ALI in rats, and these effects are partly responsible for the inhibition of ERK activation by LPS.     

Construction of a New Zealand rabbit model of diabetic nephropathy

AIM: To establish a method to produce animal model of diabetic nephropathy (DN) in New Zealand rabbits using alloxan (ALX). METHODS: Twenty male New Zealand rabbits were randomly divided into 2 groups: 8 rabbits in control group were fed with conventional feed such as buffer solution;12 in diabetes milleuts (DM) group were fed with high-sugar and high-fat diet (conventional feed plus 5% sucrose, 5% pork fat and 1% cholesterol) for 2 weeks, then intravenous injection of ALX at 50 mg/kg was given, and another dosage of 100 mg/kg ALX was intravenously injected after 48 h. The levels of blood glucose, blood cholesterol, triglyceride, urine microalbumin, and serum creatinine were detected before and 12 weeks after the processes of modeling. After 12 weeks, the rabbits were sacrificed,and the kidneys were taken for pathological examination. RESULTS: The successful model of DN in the rabbits had the characteristics of high blood glucose, abundant urine microalbumin (P<0.01), and corresponding morphological and functional changes of the kidneys. CONCLUSION: Rabbits with high-sugar and high-fat diet plus twice intravenous injection of ALX at a total dose of 150 mg/kg (50 mg/kg and 100 mg/kg, respectively) shows low mortality and stable diabetes mellitus state. This method induces a typical DN model in 12 weeks.     

Comparison of immune-related pain induced by antigen-special complex with inflammatory pain induced by formalin in rats

AIM: To investigate the difference between immune-related pain induced by antigen-special complex and inflammatory pain induced by formalin, and to observe the differential expression of p38 mitogen-activated protein kinase in spinal cord. METHODS: Thirty adult health SD rats were randomly divided into control group, formalin group and immune complex group (10 rats in each group). After the baseline tests were finished, 5 rats in each group underwent intrathecal administration of p38 MAPK inhibitor SB203580. The right hindpaw of the rats were injected with PBS, formalin or rat IgG immune complex. The thickness of hindpaw and pain behaviors were observed at time points of 0 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 h after injection. The expression levels of total and activated p38 MAPK in spinal cord were determined by Western blotting analysis. RESULTS: The rats in formalin group showed significant nociceptive behaviors immediately, such as licking foot, and limping with highly swollen foot which could touch the ground. The pain threshold was decreased rapidly 30 min after injection and alleviated after then. The pain threshold of the rats in immune complex group obviously decreased 4 h after injection without red swollen hindpaw. The expression of activated p-p38 MAPK in spinal cord in formalin group was significantly higher than that in immune complex group and control group (P<0.01). No statistic difference of p-p38 expression between immune complex group and control group, also no significant effects of SB203580 on pain behaviors in immune complex group were observed. CONCLUSION: Activated p38 MAPK contributes to the pathogenesis of inflammatory pain, but not to the pathogenesis of immune-related pain. The mechanism of immune-related pain is different from inflammatory pain induced by formalin.     

Minocycline inhibits rat ocular inflammation induced by lipopolysaccharide

AIM: To investigate the inhibitory effect of minocycline on the ocular inflammation induced by lipopolysaccharide in the rats. METHODS: Experimental endophthalmitis was induced in Sprague-Dawley rats by a single intravitreal injection of lipopolysaccharide (LPS of Escherichia coli, 1 μg). Minocycline (45 mg/kg) was administered intraperitoneally 12 h before and immediately after LPS injection and then every 24 h for 3 d. At 6, 12, 24, 48 and 72 h after LPS injection, eyes were graded daily for signs of clinical inflammation by slit lamp examination. The inflammatory cells were counted on histological sections. Vitreous was removed for cytokine analysis using standard enzyme-linked immunosorbent assay. Protein concentration in aqueous humor was determined.RESULTS: At all time points after LPS injection, significant clinical inhibition of ocular inflammation in the eye was observed in minocycline-treated rats. Quantitative analysis of ocular tissues revealed a significant decrease in infiltrated leukocytes ［(1 182.63±191.15) cells/eye in endophthalmitis group and (291.50±63.77) cells/eye in minocycline-treated group at 24 h postinjection, P<0.01］. Treatment with minocycline decreased tumor necrosis factor-α levels in vitreous ［(931.17±99.81)ng/L in endophthalmitis group and (353.02±71.67)ng/L in minocycline-treated group at 24 h postinjection, P<0.01］. Protein concentration in aqueous humor was decreased also, but the differences between the two groups at each time point was small and not significant (P>0.05).CONCLUSION: Minocycline treatment inhibits LPS-induced ocular inflammation with inhibition of tumor necrosis factor-α release and leukocytes infiltration. These results confirm the role of the tumor necrosis factor-α pathway and leukocytes infiltration in the pathogenesis of LPS-induced endophthalmitis, suggesting that minocycline may represent an attractive candidate for the therapeutic management of endophthalmitis.     

Puerarin alleviates radicular pain caused by lumbar disc herniation by inhibiting spinal glial cell activation and inflammatory response

AIM:To investigate the role of Chinese medical herb Radix Puerariae extract puerarin in a rat model of radicular pain caused by lumbar disc herniation (RAPLDH) and to explore the possible mechanism involving spinal glial cell activation and inflammatory response. METHODS:The rat model of RAPLDH was induced by autologous nucleus pulposus (NP) implantation. The rats in sham group received the same operation procedure except NP implantation. Puerarin injection at different doses (50, 100 and 150 mg/kg) was delivered intraperitoneally 1 h before surgery, and once daily for 7 d. Mechanical paw withdrawal threshold (PWT) test was employed for assessing pain behaviors. Spinal microglia and astrocyte activation was evaluated by immunofluorescence staining of relevant specific markers. The expression of pro-and anti-inflammatory cytokines in spinal dorsal horn was measured by ELISA. RESULTS:The rats with NP implantation showed long-lasting pain behaviors, characterized by decrease in PWT from day 3 to day 14 after surgery. Compared with vehicle group, puerarin at doses of 100 mg/kg and 150 mg/kg significantly increased PWT of the rats with NP implantation. Puerarin significantly reduced the expression of spinal microglia marker ionized calcium-binding adaptor molecule 1 and astrocyte marker glial fibrillary acidic protein (P<0.01). Puerarin also decreased spinal expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6, and increased anti-inflammatory cytokine interleukin-10 (P<0.01). CONCLUSION:Puerarin alleviate RAPLDH by inhibiting spinal glial cell activation and inflammatory response.     

Neuroprotective effect of L-serine on rats with permanent middle cerebral artery occlusion

AIM: To investigate the neuroprotective effect, therapeutic dosage and time window of L-serine against permanent cerebral injury in rats. METHODS: Permanent middle cerebral artery occlusion (pMCAO) was induced in the rats to determine the efficacy of L-serine (ip) by neurological evaluation, TTC staining and Nissl staining.L-serine was used at different doses (56 mg/kg, 168 mg/kg and 504 mg/kg) and for different time periods (1 h, 3 h, 6 h, 12 h and 24 h after pMCAO). Aminooxyacetic acid (AOAA), an inhibitor of serine racemase, was used to alter the efficacy of L-serine. Laser Doppler perfusion monitor was used to observe the regional cerebral blood flow (rCBF) in the ischemic cerebral cortex under the condition with or without L-serine treatment. RESULTS: Treatment with L-serine at doses of 168 mg/kg and 504 mg/kg at time point of 3 h after pMCAO greatly decreased the neurological deficit score and infarct volume,and attenuated the loss of hippocampal CA1 neuronal cells. In the observation of therapeutic time window, L-serine displayed a significant neuroprotective effect if used within 6 h after pMCAO, but did not exert any notable effect if used over 12 h after pMCAO. AOAA hardly changed the effect of L-serine. L-serine treatment notably raised rCBF in the area of ischemic cerebral cortex when it was injected 30 min after pMCAO. However, strychnine, an antagonist of strychnine-sensitive glycine receptor, did not alter this effect of L-serine. CONCLUSION: L-serine has neuroprotective effect on permanent ischemic brain injury in rats if administered early and sufficiently by augmentation of rCBF in the ischemic cerebral cortex.