Connecting cellular mechanisms and extracellular vesicle cargo in traumatic brain injury

Traumatic brain injury is followed by a cascade of dynamic and complex events occurring at the cellular level. These events include: diffuse axonal injury, neuronal cell death, blood-brain barrier break down, glial activation and neuroinflammation, edema, ischemia, vascular injury, energy failure, and peripheral immune cell infiltration. The timing of these events post injury has been linked to injury severity and functional outcome. Extracellular vesicles are membrane bound secretory vesicles that contain markers and cargo pertaining to their cell of origin and can cross the blood-brain barrier. These qualities make extracellular vesicles intriguing candidates for a liquid biopsy into the pathophysiologic changes occurring at the cellular level post traumatic brain injury. Herein, we review the most commonly reported cargo changes in extracellular vesicles from clinical traumatic brain injury samples. We then use knowledge from animal and in vitro models to help infer what these changes may indicate regrading cellular responses post traumatic brain injury. Future research should prioritize labeling extracellular vesicles with markers for distinct cell types across a range of timepoints post traumatic brain injury.

Probiotic microorganisms affect the reproductive and nervous systems of male rats treated with acrylamide

Objective:To evaluate the protective effects of probiotic microorganisms on the reproductive and nervous systems of male rats treated with acrylamide.Methods:Thirty-two rats were randomly divided into 4 groups and received normal saline through gavage(control),acrylamide 20 mg/kg body weight,acrylamide plus probiotic microorganisms(Lactobacillus acidophilus,Lactobacillus casei,Lactobacillus bulgaricus,Lactobacillus rhamnosus,Bifidobacterium breve,Bifidobacterium infantis,Streptococcus thermophilus and fructooligosaccharides,all mixed in sachets)20 or 200 mg/kg body weight,respectively.After 30 days,the testis,prostate,seminal vesicle and cerebellum were removed,fixed and stained with hematoxylin-eosin(H&E).The Johnsen score was used to classify spermatogenesis.Cavalieri's principle method was used to evaluate the total volume(in mm3)of the testes.The number of each intratubular cell type as well as intertubular Leydig cells in whole samples was measured using the physical dissector counting techniques.Stereological analysis and the grids were used to determine the volume of cerebellar layers as well as the Purkinje cell number.Results:The testis weight decreased significantly in the acrylamide-treated group compared to the other groups(P<0.001).The number of spermatogonia,spermatocytes,spermatids and Leydig cells in the acrylamide-treated group were significantly less compared to the control group(P<0.05),while they were increased significantly in the acrylamide+200 mg/kg probiotic group(P<0.05;P<0.01).The mean Johnsen score in the acrylamide-treated group was lower than in the control group(P<0.001).Acrylamide-induced changes including congestion,vacuolization in the secretory epithelial cells,and epithelial rupture were observed in the prostate and seminal vesicle.The volumes of cerebellar layers were decreased in the acrylamide group compared to the control group while recovered in both probiotic treated groups.Conclusions:Probiotic microorganisms alleviate acrylamide-induced toxicities against the reproductive and cerebellar tissues in rats.

Attenuation of MET-mediated migration and invasion in hepatocellular carcinoma cells by SOCS1

AIM To investigate the role of suppressor of cytokine signaling 1(SOCS1)in regulating MET-mediated invasive potential of hepatocellular carcinoma(HCC)cells.METHODSStable derivatives of mouse(Hepa1-6)and human(hep3B,Hep G2)HCC cell lines expressing SOCS1or control vector were evaluated for their ability to migrate towards hepatocyte growth factor(HGF)in the transwell migration assay,invade extracellular matrix in response to HGF stimulation in a 3-D invasion assay by confocal microscopy,and to undergo anchorageindependent proliferation in semisolid agar.Following intravenous and intrasplenic inoculation into NOD.scid.gamma mice,the ability of Hepa cells to form othotopic tumors was evaluated.Following HGF stimulation of Hepa and Hep3B cells,expression of proteins implicated in epithelial-to-mesenchymal transition was evaluated by western blot and qR T-PCR.RESULTS SOCS1 expression in mouse and human HCC cells inhibited HGF-induced migration through matrigel.In the 3-D invasion assay,HGF stimulation induced invasion of HCC cells across type-Ⅰcollagen matrix,and SOCS1expression significantly reduced the depth of invasion.SOCS1 expression also reduced the number and size of colonies formed by anchorage-independent growth in semisolid agar.Following intravenous inoculation,control Hepa cell formed large tumor nodules that obliterated the liver whereas the SOCS1-expressing Hepa cells formed significantly smaller nodules.Tumors formed by SOCS1-expressing cells showed reduced phosphorylation of STAT3 and ERK that was accompanied by reduced levels of MET protein expression.HGF stimulated Hepa cells expressing SOCS1 showed increased expression of E-cadherin and decreased expression of EGR1,SNAI1and ZEB1.Comparable results were obtained with Hep3B cells.SOCS1 expressing HCC cells also showed reduced levels of EGR1 and SNAI1 transcripts.CONCLUSION Our findings indicate that loss of SOCS1-dependent control over epithelial-to-mesenchymal transition may contribute to MET-mediated migration,invasion and metastatic growth of HCC.

PRIMARY CELL CULTURE FOR EMBRYONIC CARDIAC MYOCYTES OF MEXICAN AXOLOTL AND DISTRIBUTION OF DESMIN AND VIMENTIN

Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 39 were cultured for 1-14 days. Myocytes showed spontaneous contractions (15-30 beats/min) after 48-72 hours in culture, round shape and large irregular projections. Desmin and vimentin were observed in the cultured myocytes by means of immunofluorescent staining in combination with immunofluorescent microscopy. Immunofluorescent staining of the cultured cardiac myocytes after different lengths of time in culture(3,6,9 days) showed that vimentin staining was stronger than desmin staining during the early stages of culture (3 days). The myocytes exhibited various forms of staining, including parallel lines and interconnected networks. Some lines showed regular striation; most of the myofibrils were arranged in parallel arrays along the cell's long axis. Both desmin and vimentin in the cell appeared to encirele the Z lines and to link myofibrils laterally at the Z lines.

A tale of motor neurons and CD4＋ T cells： moving forward by looking back

Amyotrophic lateral sclerosis （ALS） is a fatal progressive disorder characterized by the selective degeneration of motor neurons （MN）. The impact of peripheral immune status on disease progression and MN survival is becoming increasingly recognized in the ALS research field. In this review, we briefly discuss findings from mouse models of peripheral nerve injury and immunodeficiency to understand how the immune system regulates MN survival. We extend these observations to similar studies in the widely used superoxide dismutase 1 （SOD1） mouse model of ALS. Last, we present future hypotheses to identify potential causative factors that lead to immune dysregulation in ALS. The lessons from preceding work in this area offer new exciting directions to bridge the gap in our current understanding of immune mediated neuroprotection in ALS.

Long-term administration of scopolamine interferes with nerve cell proliferation, differentiation and migration in adult mouse hippocampal dentate gyrus, but it does not induce cell death

Long-term administration of scopolamine, a muscarinic receptor antagonist, can inhibit the survival of newly generated cells, but its effect on the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus remain poorly understood. In this study, we used immunohistochemistry and western blot methods to weekly detect the biological behaviors of nerve cells in the hippocampal dentate gyrus of adult mice that received intraperito- neal administration of scopolamine for 4 weeks. Expression of neuronal nuclear antigen （NeuN; a neuronal marker） and Fluoro-]ade B （a marker for the localization of neuronal degeneration） was also detected. After scopolamine treatment, mouse hippocampal neurons did not die, and Ki-67 （a marker for proliferating cells）-immunoreactive cells were reduced in number and reac hed the lowest level at 4 weeks. Doublecortin （DCX; a marker for newly generated neurons）-im- munoreactive cells were gradually shortened in length and reduced in number with time. After scopolamine treatment for 4 weeks, nearly all of the 5-bromo-2＇-deoxyuridine （BrdU）-labeled newly generated cells were located in the subgranular zone of the dentate gyrus, but they did not migrate into the granule cell layer. Few mature BrdU/NeuN double-labeled cells were seen in the subgranular zone of the dentate gyrus. These findings suggest that long-term administration of scopolamine interferes with the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus, but it does not induce cell death.

Dual regulatory role for phosphatase and tensin homolog in specification of intestinal endocrine cell subtypes

AIM:To investigate the impact of phosphatase and tensin homolog(Pten) in the specification of intestinal enteroendocrine subpopulations.METHODS:Using the Cre/loxP system,a mouse with conditional intestinal epithelial Pten deficiency was generated.Pten mutant mice and controls were sacrificed and small intestines collected for immunofluorescence and quantitative real-time polymerase chain reaction.Blood was collected on 16 h fasted mice by cardiac puncture.Enzyme-linked immunosorbent assay was used to measure blood circulating ghrelin,somatostatin(SST) and glucose-dependent insulinotropic peptide(GIP) levels.RESULTS:Results show an unexpected dual regulatory role for epithelial Pten signalling in the specification/differentiation of enteroendocrine cell subpopulations in the small intestine.Our data indicate that Pten positively regulates chromogranin A(CgA) expressing subpopulations,including cells expressing secretin,ghrelin,gastrin and cholecystokinin(CCK).In contrast,Pten negatively regulates the enteroendocrine subtype specification of non-expressing CgA cells such as GIP and SST expressing cells.CONCLUSION:The present results demonstrate that Pten signalling favours the enteroendocrine progenitor to specify into cells expressing CgA including those producing CCK,gastrin and ghrelin.

The dual role of microglia in ischemic stroke and its modulation via extracellular vesicles and stem cells

Stem cell-based therapies and extracellular vesicle(EV)treatment have demonstrated significant potential for neuroprotection against ischemic stroke.Although the neuroprotective mechanisms are not yet fully under-stood,targeting microglia is central to promoting neuroprotection.Microglia are the resident immune cells of the central nervous system.These cells are crucial in the pathogenesis of ischemic stroke.They respond rapidly to the site of injury by releasing pro-inflammatory cytokines,phagocytizing dead cells and debris,and recruiting peripheral immune cells to the ischemic area.Although these responses are essential for clearing damage and initiating tissue repair,excessive or prolonged microglial activation can exacerbate brain injury,leading to secondary neuroinflammation and neurodegeneration.Moreover,microglia exhibit a dynamic range of activation states with the so-called M1 pro-inflammatory and M2 anti-inflammatory phenotypes,representing the two ends of the spectrum.The delivery of both EVs and stem cells modulates microglial activation,suppressing pro-inflammatory genes,influencing the expression of transcription factors,and altering receptor expression,ultimately contributing to neuroprotection.These findings underscore the importance of understanding the complex and dynamic role of microglia in the development of effective neuroprotective strategies to reduce the effects of ischemic stroke.In this review,we examine the current state of knowledge regarding the role of microglia in ischemic stroke,including their molecular and cellular mechanisms,activation states,and interactions with other cells.We also discuss the multifaceted contributions of microglia to stem cell-and EV-based neuroprotection during an ischemic stroke to provide a comprehensive understanding of microglial functions and their potential implications in stroke therapies.

Adult human neural stem cell therapeutics: Current developmental status and prospect

Over the past two decades, regenerative therapies using stem cell technologies have been developed for various neurological diseases. Although stem cell therapy is an attractive option to reverse neural tissue damage and to recover neurological deficits, it is still under development so as not to show significant treatment effects in clinical settings. In this review, we discuss the scientific and clinical basics of adult neural stem cells(a NSCs), and their current developmental status as cell therapeutics for neurological disease. Compared with other types of stem cells, a NSCs have clinical advantages, such as limited proliferation, inborn differentiation potential into functional neural cells, and no ethical issues. In spite of the merits of a NSCs, difficulties in the isolation from the normal brain, and in the in vitro expansion, have blocked preclinical and clinical study using a NSCs. However, several groups have recently developed novel techniques to isolate and expand a NSCs from normal adult brains, and showed successful applications of a NSCs to neurological diseases. With new technologies for a NSCs and their clinical strengths, previous hurdles in stem cell therapies for neurological diseases could be overcome, to realize clinically efficacious regenerative stem cell therapeutics.

Prosaposin ablation inactivates the MAPK and Akt signaling pathways and interferes with the development of the prostate gland

<abstract>The recent development of a prosaposin -/- mouse model has allowed the investigation of the role of prosaposin in the development of the male reproductive organs. A morphometric analysis of the male reproductive system of 37 days old mice revealed that prosaposin ablation produced a 30 % reduction in size and weight of the testes, 37 % of the epididymis, 75 % of the seminal vesicles and 60 % of the prostate glands. Light microscopy (LM) showed that smaller testis size from homozygous mutant mice was associated with reduced spermiogenesis. Both, dorsal and ventral lobules of the prostate glands were underdeveloped in the homozygous mutant. LM analysis also showed that prostatic alveoli were considerably smaller and lined by shorter epithelial cells in the homozygous mutant. Smaller tubular diameter and shorter undifferentiated epithelial cells were also observed in seminal vesicles and epididymis. In the efferent ducts of the homozygous mutant mice, the epithelium was composed exclusively of ciliated cells in contrast to the heterozygotes, which showed the presence of nonciliated cells. Radioimmunoassays demonstrated that testosterone levels were normal or higher in mice with the inactivated prosaposin gene. Immunostaining of prostate sections with an anti-androgen receptor antibody showed that the epithelial cells lining the alveoli express androgen receptor in both the heterozygous and homozygous tissue. Similarly, sections immunostained with antibodies to the phosphorylated MAPKs and Akts strongly reacted with tall prostatic secretory cells in prostate from heterozygous mouse. On the other hand, the epithelial cells in the homozygous prostate remained unstained or weakly stained. These findings demonstrate that inactivation of the prosaposin gene affected the development of the prostate gland and some components of the MAPK pathway. 63 )

Expression of von Hippel-Lindau tumor suppressor and tumor-associated carbonic anhydrases IX and XII in normal and neoplastic colorectal mucosa

AIM:To analyze possible relationships between CA IX/ CA XII and pVHL expression in normal and neoplastic colorectal mucosa. METHODS: Immunohistochemical staining of 42 tissue specimens obtained from 17 cancer patients was performed to evaluate the distribution and semi-quantitatively assess the levels of CA IX, CA XII and pVHL. VHL mRNAs from 14 fresh-frozen tumors was amplified by RT-PCR and subjected to sequencing. CA9 and G412mRNA levels were analyzed by semi-quantitative RT-PCR in comparison with VEGFas an indicator of hypoxia that uncouples the pVHL control. RESULTS: Tumor tissues were associated with a borderline increase of CA IX staining signal and slight but significant decrease of CA XII immunoreactivity, whereas no association was found for pVHL. Sequence analysis of RT-PCR-amplified VHL mRNAs revealed no deletions/ mutations, suggesting that they were VHL-competent. We did not observe any correlation between pVHL and CA IX/CA XII proteins as well as between MEGFand CA9 mRNAs, but the tumor-associated changes in mRNA levels of VEGFand CA12 showed a significant inverse relationship. CONCLUSION: Our results indicate that CA9and CA12 are regulated by different intratumoral factors and that lack of apparent relationship between the levels of CA IX/CA XII and pVHL cannot be fully assigned to uncoupling of negative regulatory function of pVHL by tumor hypoxia signified by induced VEGF transcription. The interplay between the functional pVHL and CA IX/CA XII in colorectal tumors seems rather complex and is not evident merely at the expression levels.

Exercise-induced irisin in bone and systemic irisin administration reveal new regulatory mechanisms of bone metabolism

Irisin is a polypeptide hormone derived from the proteolytic cleavage of fibronectin-type III domain- containing 5 （FNDC5） protein. Once released to circulation upon exercise or cold exposure, irisin stimulates browning of white adipose tissue （WAT） and uncoupling protein I （UCP1） expression, leading to an increase in total body energy expenditure by augmented UCPl-mediated thermogenesis. It is currently unknown whether irisin is secreted by bone upon exercise or whether it regulates bone metabolism in vivo. In this study, we found that 2 weeks of voluntary wheel-running exercise induced high levels of FNDC5 messenger RNA as well as FNDC5/irisin protein expression in murine bone tissues. Increased immunoreactivity due to exercise-induced FNDC5/irisin expression was detected in different regions of exercised femoral bones, including growth plate, trabecular bone, cortical bone, articular cartilage, and bone-tendon interface. Exercise also increased expression of osteogenic markers in bone and that of UCP1 in WAT, and led to bodyweight loss. Irisin intraperitoneal （IP） administration resulted in increased trabecular and cortical bone thickness and osteoblasts numbers, and concurrently induced UCP1 expression in subcutaneous WAT. Lentiviral FNDC5 IP administration increased cortical bone thickness. In vitro studies in bone cells revealed irisin increases osteoblastogenesis and mineralization, and inhibits receptor activator of nuclear factor-kB ligand （RANKL）- induced osteoclastogenesis. Taken together, our findings show that voluntary exercise increases irisin production in bone, and that an increase in circulating irisin levels enhances osteogenesis in mice.

Histopathological studies of acute and chronic effects of Calliandra portoricensis leaf extract on the stomach and pancreas of adult Swiss albino mice

Objective:To evaluate the consequence of oral administration of Calliandra portoricensis(C. portoricensis) leaf extract on the stomach and pancreas in Swiss albino mice.Methods:Three, groups of mice(B,C and D) were treated with 4 mg/kg of C.portoricensis extract.Croup A was the control and received an equivalent volume of distilled water.Group B received C.portoricensis leaf extract for 7 days.Croup C received C.portoricensis leaf extract for 14 days,and Croup D received C.portoricensis leaf extract for 28 days.At different stages in the study,the mice were sacrificed and the stomach and pancreas were excised and fixed in 10%formol saline for histological analysis.Results:The result showed a normal microstructural outline in groups B and C as compared with the control.However,animals in group D showed disorganization of the mucosa and discontinuation of epithelial lining of the stomach while the islets of Langerans in the pancreas were at various degree of degeneration as compared with the control mice. Conclusions:The present finding suggests that chronic administration(28 days as seen in this study) of C.portoricensis leaf extract may inhibit the proper function of the stomach and pancreas.

Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

Bone morphogenetic proteins （Bmp） are well-known to induce bone formation following chondrogenesis, but the direct role of Bmp signaling in the osteoblast lineage is not completely understood. We have recently shown that deletion of the receptor Bmprla in the osteoblast lineage with Dmpl-Cre reduces osteoblast activity in general but stimulates proliferation of preosteoblasts specifically in the cancellous bone region, resulting in diminished periosteal bone growth juxtaposed with excessive cancellous bone formation. Because expression of sclerostin （SOST）, a secreted Wnt antagonist, is notably reduced in the Bmprla- deficient osteocytes, we have genetically tested the hypothesis that increased Wnt signaling might mediate the increase in cancellous bone formation in response to Bmprla deletion. Forced expression of human SOST from a Dmpl promoter fragment partially rescues preosteoblast hyperproliferation and cancellous bone overgrowth in the Bmprla mutant mice, demonstrating functional interaction between Bmp and Wnt signaling in the cancellous bone compat^a-tent. To test whether increased Wnt signaling can compensate for the defect in periosteal growth caused by Bmprla deletion, we have generated compound mutants harboring a hyperactive mutation （A214V） in the Wnt receptor Lrp5. However, the mutant Lrp5 does not restore periosteal bone growth in the Bmprla-deficient mice. Thus, Bmp signaling restricts cancellous bone accrual partly through induction of SOST that limits preosteoblast proliferation, but promotes periosteal bone growth apparently independently of Wnt activation.

Orchestration of occludins, claudins, catenins and cadherins as players involved in maintenance of the blood-epididymal barrier in animals and humans

Although spermatozoa are formed during spermatogenesis in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical spermatozoal functions are acquired in the epididymis where a specific luminal environment is created by the blood-epididymal barrier; proteins secreted by epididymal principal cells bind to maturing spermatozoa and regulate the maturational process of the spermatozoa. In the epididymis, epithelial cell-cell interactions are mediated by adhering junctions, necessary for cell adhesion, and by tight junctions, which form the blood-epididymal barrier. The regulation of these cellular junctions is thought to represent a key determinant in the process of sperm maturation within the epididymis. Tight junctions between adjacent principal cells permit the formation of a specific microenvironment in the lumen of the epididymis that is essential for sperm maturation. Although we have made significant progress in understanding epididymal function and the blood-epididymal barrier, using animal models, there is limited information on the human epididymis. If we are to understand the normal and pathological conditions attributable to human epididymal function, we must clearly establish the physiological, cellular and molecular regulation of the human epididymis, develop tools to characterize these functions and develop clinical strategies that will use epididymal functions to improve treatment of infertility. （Asian J Androl 2007 July; 9： 463- 475）

Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells

Mutations in the liver/bone/kidney alkaline phosphatase(Alpl) gene cause hypophosphatasia(HPP) and early-onset bone dysplasia,suggesting that this gene is a key factor in human bone development. However, how and where Alpl acts in bone ageing is largely unknown. Here, we determined that ablation of Alpl induces prototypical premature bone ageing characteristics, including bone mass loss and marrow fat gain coupled with elevated expression of p16INK4A(p16) and p53 due to senescence and impaired differentiation in mesenchymal stem cells(MSCs). Mechanistically, Alpl deficiency in MSCs enhances ATP release and reduces ATP hydrolysis. Then, the excessive extracellular ATP is, in turn, internalized by MSCs and causes an elevation in the intracellular ATP level, which consequently inactivates the AMPKα pathway and contributes to the cell fate switch of MSCs. Reactivating AMPKα by metformin treatment successfully prevents premature bone ageing in Alpl+/-mice by improving the function of endogenous MSCs.These results identify a previously unknown role of Alpl in the regulation of ATP-mediated AMPKα alterations that maintain MSC stemness and prevent bone ageing and show that metformin offers a potential therapeutic option.

Expression patterns of transforming growth factor-beta and its receptors in gastric mucosa of patients with refractory gastric ulcer

AIM: Transforming growth factor-β (TGF-β) plays a regulatory role in tissue repair. In a previous study, we found that TGF-β and its receptors were expressed in gastric mucosa of patients with well-healed gastric ulcers, as demonstrated by immunohistochemistry. To further characterize the role of TGF-β and its receptors in repairing gastric ulcers, we investigated the expression patterns of TGF-β and its receptors in gastric mucosa by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR).METHODS: Seventy-four patients with endoscopically proven gastric ulcers were eligible for participation in this study. All patients had routine biopsies on initial endoscopy and were then treated for 12 wk with an H2 blocker. Repeat endoscopy was then performed. There were 8 patients with poorly healed ulcers, and biopsies were taken from the margin of the residual ulcers. These tissue samples, along with biopsy of gastric mucosa near the original ulcers from 8 randomly selected patients with well-healed ulcers were examined for TGF-β and TGF-β receptor Ⅱ mRNA by RT-PCR and in situ hybridization, as well as immunohistochemistry.RESULTS: TGF-β and TGF-β receptor Ⅱ were strongly expressed in tissues from patients with well-healed ulcers.Four of the 8 patients with poor healing had low or absent expression of TGF-β or TGF-β receptor Ⅱ mRNA. All cases positive by RT-PCR assay were confirmed by in situ hybridization as well as immunohistochemistry.CONCLUSION: It is suggested that TGF-β and its receptors are important for gastric ulcer healing. These results may have implications for further investigation of the healing process and in predicting response to therapy.

Effects of protein deprivation and re-feeding on P2X_2 receptors in enteric neurons

AIM:To investigate the effects of malnutrition and refeeding on the P2X2 receptor,nitric oxide synthase(NOS),calretinin,calbindin and choline acetyltransferase(ChAT) in neurons of the rat ileum.METHODS:We analyzed the co-localization,numbers and sizes of P2X2-expressing neurons in relation to NOS-immunoreactive(IR),calbindin-IR,ChAT-IR,and calretinin-IR neurons of the myenteric and submucosal plexus.The experimental groups consisted of:(1) rats maintained on normal feed throughout pregnancy until 42 d post-parturition(N);(2) rats deprived of protein throughout pregnancy and 42 d post-parturition(D);and(3) rats undernourished for 21 d post-parturition and then given a protein diet from days 22 to 42(DR).The myenteric and submucosal plexuses were evaluated by double labeling by immunohistochemical methods for P2X2 receptor,NOS,ChAT,calbindin and calretinin.RESULTS:We found similar P2X2 receptor immunoreactivity in the cytoplasm and surface membranes of myenteric and submucosal neurons from the N,D and DR groups.Double labeling of the myenteric plexus demonstrated that approximately 100% of NOS-IR,calbindin-IR,calretinin-IR and ChAT-IR neurons in all groups also expressed the P2X2 receptor.In the submucosal plexus,the calretinin-IR,ChAT-IR and calbindin-IR neurons were nearly all immunoreactive for the P2X2 receptor.In the myenteric plexus,there was a 19% increase in numbers per cm2 for P2X2 receptor-IR neurons,64% for NOS-IR,84% for calretinin-IR and 26% for ChAT-IR neurons in the D group.The spatial density of calbindin-IR neurons,however,did not differ among the three groups.The submucosal neuronal density increased for calbindin-IR,calretinin-IR and ChAT-IR neurons.The average size of neurons in the myenteric plexus neurons in the D group was less than that in the controls and,in the re-fed rats;there was a 34% reduction in size only for the calretinin-IR neurons.CONCLUSION:This work demonstrates that expression of the P2X2 receptor is present in inhibitory,intrinsic primary afferent,cholinergic secretomotor and vasomotor neurons.Undernutrition affected P2X2 receptor expression in the submucosal plexus,and neuronal and size.These changes were rescued in the re-fed rats.

Some of the experimental and clinical aspects of the effects of the maternal diabetes on developing hippocampus

Diabetes mellitus during pregnancy is associated with an increased risk of multiple congenital anomalies in progeny.There are sufficient evidence suggesting that the children of diabetic women exhibit intellectual and behavioral abnormalities accompanied by modification of hippocampus structure and function.Although,the exact mechanism by which maternal diabetes affects the developing hippocampus remains to be defined.Multiple biological alterations,including hyperglycemia,hyperinsulinemia,oxidative stress,hypoxia,and iron deficiency occur in pregnancies with diabetes and affect the development of central nervous system(CNS) of the fetus.The conclusion from several studies is that disturbance in glucose and insulin homeostasis in mothers and infants are major teratogenic factor in the development of CNS.Insulin and Insulin-like growth factor-1(IGF-1) are two key regulators of CNS function and development.Insulin and IGF-1 receptors(IR and IGF1 R,respectively) are distributed in a highly specific pattern with the high density in some brain regions such as hippocampus.Recent researches have clearly established that maternal diabetes disrupts the regulation of both IR and IGF1 R in the hippocampus of rat newborn.Dissecting out the mechanisms responsible for maternal diabetes-related changes in the development of hippocampus is helping to prevent from impaired cognitive and memory functions in offspring.

Locomotor analysis identifies early compensatory changes during disease progression and subgroup classification in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a motoneuron degenerative disease that is challenging to diagnose and presents with considerable variability in survival.Early identification and enhanced understanding of symptomatic patterns could aid in diagnosis and provide an avenue for monitoring disease progression.Use of the m SOD1 G93 A mouse model provides control of the confounding environmental factors and genetic heterogeneity seen in amyotrophic lateral sclerosis patients,while investigating underlying disease-induced changes.In the present study,we performed a longitudinal behavioral assessment paradigm and identified an early hindlimb symptom,resembling the common gait abnormality foot drop,along with an accompanying forelimb compensatory mechanism in the m SOD1 G93 A mouse.Following these initial changes,m SOD1 mice displayed a temporary hindlimb compensatory mechanism resembling an exaggerated steppage gait.As the disease progressed,these compensatory mechanisms were not sufficient to sustain fundamental locomotor parameters and more severe deficits appeared.We next applied these initial findings to investigate the inherent variability in B6 SJL m SOD1 G93 A survival.We identified four behavioral variables that,when combined in a cluster analysis,identified two subpopulations with different disease progression rates：a fast progression group and a slow progression group.This behavioral assessment paradigm,with its analytical approaches,provides a method for monitoring disease progression and detecting m SOD1 subgroups with different disease severities.This affords researchers an opportunity to search for genetic modifiers or other factors that likely enhance or slow disease progression.Such factors are possible therapeutic targets with the potential to slow disease progression and provide insight into the underlying pathology and disease mechanisms.