Neuroprotective effect of vitamin C against PTZ induced apoptotic neurodegeneration in adult rat brain.

The present study was designed to observe the effect of PTZ on expression of caspsae-3, and to evaluate the neuroprotective role of vitamin C (vit-C) against PTZ-induced apoptotic neurodegeneration in adult rat brain. We observed that administration of a single conclusive dose of pentylenetetrazol (PTZ 50mg/kg) in adults rats induced epileptic seizure and increased activation of caspase-3 and caused neuronal death. Further, rats were injected with vit-C (250 mg/kg) 30 min before PTZ injection. The protective effect of vit-C against PTZ-induced apoptotic neurodegeneration in adult rat brain was observed using Western blot analysis and Nissl staining. The results showed that conclusive dose of PTZ-induced seizure, increased expression of caspase-3 and neuronal apoptosis in adult rat brain. Whereas, the pretreatment of vit-C along with PTZ showed significantly decreased expression of caspase-3 as compare to control group. Finally, our results indicated that vit-C can prevent some of the deleterious effect of seizure and neuronal degeneration induced by PTZ in adult rat brain.

Lithium ameliorates lipopolysaccharide-induced neurotoxicity in the cortex and hippocampus of the adult rat brain.

Lithium an effective mood stabilizer, primary used in the treatment of bipolar disorders, has been reported as a protective agent in various neurological disorders. In this study, we examined the neuroprotective role of lithium chloride (LiCl) against lipopolysaccharide (LPS) in the cortex and hippocampus of the adult rat brain. We determined that LiCl -attenuated LPS-induced activated toll-like receptor 4 (TLR4) signalling and significantly reduced the nuclear factor-kB (NF-KB) translation factor and various other inflammatory mediators such as interleukin-1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α). We also analyzed that LiCl significantly abrogated activated gliosis via attenuation of specific markers for activated microglia, ionized calcium-binding adaptor molecule (Iba-1) and astrocytes, glial fibrillary acidic protein (GFAP) in both the cortex and hippocampus of the adult rat brain. Furthermore, we also observed that LiCl treatment significantly ameliorated the increase expression level of apoptotic neurodegeneration protein markers Bax/Bcl2, activated caspase-3 and poly (ADP-ribose) polymerase-1 (PARP-1) in the cortex and hippocampus regions of the LPS-treated adult rat brain. In addition, the morphological results of the fluoro-jade B (FJB) and Nissl staining showed that LiCl attenuated the neuronal degeneration in the cortex and hippocampus regions of the LPS-treated adult rat brain. Taken together, our Western blot and morphological results indicated that LiCl significantly prevents the LPS-induced neurotoxicity via attenuation of neuroinflammation and apoptotic neurodegeneration in the cortex and hippocampus of the adult rat brain.

Protective effects of betaxolol in eyes with kainic acid-induced neuronal death.

In the present study, we investigated whether betaxolol, a selective beta1-adrenoceptor antagonist, has neuroprotective effect on kainic acid (KA)-induced retinal damage. Neurotoxicities were induced in adult male rats by intravitreal injection of KA (total amount, 6 nmol). To examine the neuroprotective effects of betaxolol, rats were pretreated with betaxolol topically 60 min before KA injection to the rat eyes and twice daily for 1, 3, and 7 days after KA injection. The neuroprotective effects of betaxolol were estimated by measuring the thickness of the various retinal layers, and by counting the number of choline acetyltransferase (ChAT)- and tyrosine hydroxylase (TH)-positive cells in each retinal layer. The retina is highly vulnerable to KA-induced neuronal damage. Morphometric analysis of retinal damage in KA injected eyes, the thickness of the retinal layers decreased markedly after KA injection period of both 3 and 7 days. Furthermore, the numbers of ChAT- and TH-positive cells were significantly reduced by intravitreal injection of KA. However, when two drops of betaxolol, once before KA injection and twice daily for 7 days after KA injection, were continuously administered, the reductions in the retinal thickness and the retinal ChAT- and TH-positive cells were significantly attenuated. The present study suggests that topically applied betaxolol has neuroprotective effect on the retinal cell damage due to KA-induced neurotoxicity.

Action of citicoline on rat retinal expression of extracellular-signal-regulated kinase (ERK1/2).

Citicoline is an essential endogenous intermediate in the biosynthesis of phosphatidylcholine, which acts as a therapeutic agent in models of central nervous system injury and neurodegenerative diseases. The present study investigated the effects of citicoline on extracellular-signal-regulated kinase 1/2 (ERK1/2) expression in the rat retina after kainic acid (KA) treatment. KA (6 nmol) was injected into the vitreous of the rat eyes. The animals were then injected intraperitoneally with citicoline (500 mg/kg) twice daily after the KA injection. The neuroprotective effects of citicoline were estimated by evaluating temporal changes in ERK1/2 using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL), immunoblotting and immunohistochemical techniques. The expression of phosphorylated ERK1/2 was slightly decreased after 6 h, and significantly reduced after 12 h, in the rats receiving the KA injection plus citicoline treatment. Our results demonstrated that citicoline decreased the activation of ERK1/2 due to the KA treatment, suggesting that it exerts its neuroprotective activity by reducing the concentrations of proteins involved in apoptosis.

Change in endothelial nitric oxide synthase in the rat retina following transient ischemia.

Patterns of endothelial nitric oxide synthase (eNOS) expression in retinal ischemia were studied utilizing a transient high intraocular pressure (HIOP) model. We investigated neuronal cell damage and changes in eNOS immunoreactive expression in the ischemic retina, and its relationship to the neuroprotection of betaxolol treatment after ischemic injury. Immunohistochemical staining for eNOS was performed at 3, 7, 14 and 28 days after ischemia/reperfusion. In controls, eNOS immunoreactivity was detected in retinal vessels, but was not detected in neurons. After ischemia/reperfusion, the intensity of eNOS immunoreactivity increased in both retinal vessels and the ganglion cell layer (GCL) compared with controls. eNOS-positive neurons were induced first in the inner nuclear layer (INL) 7 days after reperfusion. However, when experiments were carried out on animals that had been treated with betaxolol after ischemia/reperfusion, the intensity of eNOS immunoreactivity decreased compared to the untreated ischemic retinas. These results suggest that an increase in eNOS expression could be associated with the degenerative changes in the ischemic retina, and that betaxolol treatment appears to protect retinal tissue from ischemic damage.

Betaxolol attenuates retinal ischemia/reperfusion damage in the rat.

This study was performed to elucidate the protection afforded by post-treatment with Betoptic (0.25% betaxolol) against neuronal cell damage after ischemia/reperfusion insult in rats. Betaxolol was applied topically after the start of reperfusion and its effect was evaluated by morphometry and choline acetyltransferase immunoreactivity of retinas at 7 days after reperfusion. In non-treated eyes, the thickness of the inner plexiform layer decreased markedly after a reperfusion period of both 3 and 7 days. However, when eyes were treated with betaxolol after ischemia/reperfusion injury, both the reduction of the inner plexiform layer thickness and the retinal choline acetyltransferase immunoreactivity were significantly attenuated. These findings suggest that betaxolol is an efficient neuroprotective agent and prevents the retinal cell damage induced by ischemic injury in rats.

Expression of steroidogenic acute regulatory protein mRNA in rat placenta during mid-late pregnancy.

The production of a steroid hormone in the placenta is essential for maintaining the pregnancy and developing the fetus during gestation. In various steroidogenic tissues (including gonads and adrenal cortex), the steroidogenic-acute-regulatory protein (StAR) acutely transfers cholesterol from the outer to the inner mitochondrial membrane for rapid steroidogenesis. Although steroid hormones were synthesized in the rat placenta, the developmental expression of StAR has been poorly understood in the rat placenta during mid-late pregnancy. Therefore, the aim of the present study was to establish the expression and localization of StAR mRNA in the rat placenta during mid-late pregnancy using Northern blots and in situ hybridization. The Northern blot analysis showed that the StAR mRNA expression significantly changed as the gestation day (GD) progressed. The placental expression of StAR mRNA increased between GD 11 and 13, and then slightly decreased until term. In situ hybridization showed a strong StAR expression in giant trophoblast cells on GD 11 and 13, and a moderate expression in trophoblast and stroma cells within the villi of the labyrinth zone throughout the pregnancy. In this study, we reveal for the first time the existence of StAR mRNA in steroidogenic cells of the placenta during mid-late pregnancy. In conclusion, our results suggest that StAR may regulate steroidogenesis in the rat placenta to maintain the pregnancy and developing the fetus.

Cellular localization of pituitary adenylate cyclase-activating polypeptide in the rat testis.

Several growth factors are critical regulators of testicular development. Recently, pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to promote the differentiation and proliferation of various cell types and to stimulate the release of vascular endothelial growth factor during tissue growth. The present study aimed to evaluate the possible expression of PACAP in immature and adult rat testes by Northern blot analysis, in situ hybridization, and immunohistochemistry. PACAP transcripts were detected in both immature and adult tissues, but the level of PACAP expression depended on the age of the animal. It was expressed in the peritubular and interstitial cells of the 14-day-old testis and in seminiferous tubules of the 90-day-old-testis. It was strongly expressed in blood vessels at 14 days, moderately at 28 days and only weakly at 90 days. These results suggest that PACAP contributes to the growth and differentiation of peritubular and interstitial cells, and of blood vessels, during testicular development.

Retinal expression of clusterin in the streptozotocin-induced diabetic rat.

To assess the possible relevance of clusterin in the pathophysiology of retinopathy associated with diabetes mellitus, streptozotocin-induced diabetic rats were studied. Clusterin expression was measured in both normal and streptozotocin-induced diabetic rat retinas using Northern blotting, reverse transcription polymerase chain reaction, immunohistochemistry and Western blot analysis. The results show increased clusterin protein level and its mRNA expression 6 weeks after induction of diabetes. Clusterin was localized to the inner nuclear and ganglion cell layers of both normal and diabetic rat retinas. These data show that diabetes affects the expression of clusterin in the retina and may reflect a diabetes-induced damage and/or alterations of neural structures resulting in diabetic retinopathy.

Nitric oxide synthase expression in the transient ischemic rat retina: neuroprotection of betaxolol.

Betaxolol is a beta-adrenergic blocker but its neuroprotective action is generally thought to be due to its calcium channel blocking properties. In this study, we investigated neuronal cell damage and changes in the expression of neuronal nitric oxide synthase (nNOS) immunoreactivity in the ischemic retina and its relationship to the neuroprotection of betaxolol treatment after ischemic injury. Using the retina after ischemia, the expression of nNOS was studied by immunocytochemistry. In control retinas, two types of amacrine cells and a class of displaced amacrine cells were nNOS-labeled. After ischemia/reperfusion, the number of nNOS immunoreactive cells increased in both the ganglion cell layer and the inner nuclear layer compared to the control retinas. However, when experiments were carried out on animals that had been treated with betaxolol twice daily after ischemia/reperfusion, the number of nNOS immunoreactive cells decreased compared to the untreated ischemic retinas. These results suggest that an increase in nNOS expression could be associated with the degenerative changes in the ischemic retina, and that betaxolol treatment appears to play a role in protecting retinal tissue from ischemic damage.

Upregulation of glucose-dependent insulinotropic polypeptide and its receptor in the retina of streptozotocin-induced diabetic rats.

The pathology of diabetic retinopathy includes dilatation and beading of retinal vessels, and vascular sheathing. To gain a better understanding of the molecular events leading to diabetic retinopathy, we investigated disease-specific gene responses by screening differential expression using cDNA microarray. Male Sprague-Dawley rats were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg) or the control buffer and were maintained for 6 weeks. Total RNA extracted from the retinas of both groups was used for cDNA microarray analysis. Signals from all the spots representing hybridized DNA were quantified and compared between the normal and diabetic rat retinas. Among 1176 genes analyzed, the retinal expression of glucose-dependent insulinotropic polypeptide (GIP) was found to increase in STZ-induced diabetic rats compared to controls. GIP is a secreted protein, known to be released from the small intestine, which potentiates glucose-induced insulin secretion from the pancreas. However, the expression of GIP and its receptor (GIPR) has not been previously noted in the rat retina. To further validate the expression of GIP in the rat retina and to determine its possible role in the development of early diabetic retinopathy, we investigated its expression by RT-PCR, Northern blotting, and immunohistochemistry in normal and diabetic rat retinas. GIP mRNA and protein are not only expressed in the rat retina, but their levels are greater in the diabetic rat as compared to controls. And GIPR expression was also upregulated in the retinas of STZ-induced diabetic rats. We here demonstrate for the first time the expression of GIP and GIPR in the rat retina. And we also revealed some genetic events in the early stage of diabetic retinopathy including the de novo increment of GIP and GIPR expression in the retina.

Neuroprotective effect of citicoline against KA-induced neurotoxicity in the rat retina.

We examined whether citicoline has neuroprotective effect on kainic acid (KA)-induced retinal damage. KA (6 nmol) was injected into the vitreous of rat eyes. Rats were injected intraperitoneally with citicoline (500 mgkg-1, i.p.) twice (09:00 and 21:00) daily for 1, 3 and 7 days after KA-injection. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers. In addition, immunohistochemistry was conducted to elucidate the expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH). Morphometric analysis of retinal damage in KA-injected eyes showed a significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at the 1, 3 and 7 days after KA injection, but not in the outer nuclear layers (ONL). At 1 and 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of ChAT and TH. The immunoreactivities of ChAT and TH had almost disappeared in the retina after 7 days of KA injection. However, prolonged citicoline treatment for 7 days significantly attenuated the reduction of retinal thickness and immunoreactivities of ChAT and TH. The present study suggests that treatment with citicoline has neuroprotective effect on the retinal damage due to KA-induced neurotoxicity.

Changes in rhodopsin kinase and transducin in the rat retina in early-stage diabetes.

To establish changes in phototransduction in diabetes, the effects of high glucose on rhodopsin kinase (RK) and transducin (G(t)), as well as recoverin, were examined in the retina of STZ-induced diabetic rats. Diabetes was induced by single intraperitoneal injection of STZ (50mg/kg) to Sprague-Dawley (SD) rats and the animals were sacrificed after 6 weeks. Immunohistochemistry (IHC) and Western blot analysis were carried out using antibodies against RK and G(talpha) (alpha subunit of G(t)) in the STZ-induced diabetic retina and the control retina. The expression level of recoverin protein was also analysed. In the diabetic retina, while the expression of RK protein increased, that of G(talpha) and recoverin proteins decreased. RK immunoreactivity (IR) appeared generally in the retina, and its signal increased in the outer limiting membrane (OLM), some rod cells in the outer segment layer (OSL) and at the tip of the outer plexiform layer (OPL) in the diabetic retina. G(talpha)-IR also appeared in the OPL and in photoreceptor layer. In the diabetic retina, G(talpha)-IR significantly decreased in the OPL, indicating RK-IR increase. This study illustrates the alterations in RK, G(talpha) and recoverin in the diabetic retina that may induce dysfunctions in phototransduction even in early-stage diabetes.