Involvement of PDZ-SAP97 interactions in regulating AQP2 translocation in response to vasopressin in LLC-PK1 cells.

Arginine-vasopressin (AVP)-mediated translocation of aquaporin-2 (AQP2) protein-forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for the renal conservation of water. The type-1 PDZ-binding motif (PBM) in AQP2, "GTKA," is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remain obscure. We determined that synapse-associated protein-97 (SAP97), a membrane-associated guanylate kinase protein involved in establishing epithelial cell polarity, was an avid binding partner to the PBM of AQP2. The role of PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC-PK1 renal collecting cells by confocal microscopy and cell surface biotinylation techniques. These experiments indicated that distribution of AQP2 and SAP97 overlapped in the kidneys and LLC-PK1 cells and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. Binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of PKA, or knockdown of SAP97 inhibited AQP2 translocation as well as AVP- and forskolin-mediated phosphorylation of Ser256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that the targeting of PKA to the microdomain of AQP2 via SAP97-AQP2 interactions in association with cross-talk between two barcodes in AQP2, namely, the PBM and phospho-Ser256, plays an important role in the translocation of AQP2 in the kidney.

Integrative management of diabetic foot ulcers - A case series.

Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus and a cause of significant morbidity, mortality and healthcare expenditure. Treatment of DFU includes multimodal approach like surgical debridement, infection control, vascular assessment, dressing etc. Multidisciplinary approach towards foot care is becoming a mainstay of therapy, and even with this comprehensive approach, there is still room for improvement in DFU outcomes. Integrative management includes the adoption of various systems of treatment with standard treatment for better outcomes. In the present case series, six cases of DFU were managed with the integration of Ayurveda and allopathic treatments. The cases were managed according to the standard diabetic foot ulcer management principles like surgical debridement, insulin therapy, along with incorporation of Ayurveda procedures like Vimlapana, Prakshalana, Bandhana etc. Standard assessment of ulcers at different time points was done using the Bates- Jensen Ulcer assessment tool. All the six DFU healed with minimal scar formation and in less time, lowering the risk of further amputation. Promising results were obtained in all six cases by adopting integrated Ayurveda and allopathic treatments, which indicates the potential benefits of alternative systems of medicine.

Zinc finger protein­like 1 is a novel neuroendocrine biomarker for prostate cancer.

Prostate­derived calcitonin (CT) and its receptor induce tumorigenicity and increase metastatic potential of prostate cancer (PC). CT­inducible genes in human prostate were identified by subtraction hybridization. Among these genes, zinc finger protein like 1 (ZFPL1) protein was interesting since it was abundantly expressed in malignant prostates but was almost absent in benign prostates. ZFPL1 expression was upregulated by CT and androgens, and ZFPL1 protein was secreted by prostate tumor cells through exosomal secretion. Serum levels of ZFPL1 in cancer patients were at least 4­fold higher than those in the sera of cancer­free individuals. Cell biology of ZFPL1 suggests its localization in Golgi bodies and exosomes, and its colocalization with chromogranin A and CD44. These results suggested that ZFPL1 is secreted by tumor cells of neuroendocrine (NE)/stem cell phenotype. The knockdown of endogenous ZFPL1 in (PC) cells led to a remarkable decrease in cell proliferation, and invasion while increasing their apoptosis. As expected, the overexpression of ZFPL1 in prostate cells had an opposite effect on these functions. The knockdown of ZFPL1 in PC cells also decreased Akt phosphorylation, suggesting the actions of ZFPL1 may be mediated through the PI3K­Akt pathway. Moreover, the present results revealed that ZFPL1 is released by tumors cells of NE or androgen­independent phenotype and its serum levels are significantly higher in cancer patients, suggesting that it may serve as a blood­based non­invasive biomarker of aggressive PC.

The Etiological Profile of Global Developmental Delay at a Tertiary Care Hospital in India: An Observational Study.

Background Global developmental delay (GDD) is common and has a significant impact on affected children, families, and society. Understanding its etiology is crucial for management and prevention strategies. However, data on the etiological profile of GDD in developing countries are limited. This study aimed to identify the etiological profile of GDD at a tertiary care hospital in India. Methodology This observational study included children aged three months to five years with a developmental quotient below 70%. Data on demographics, clinical features, relevant investigations, and diagnoses were collected. Etiologies were categorized into prenatal, perinatal, postnatal, and unknown causes. Informed consent was obtained from the parents. Results A total of 52 children, with a median age of 15.5 months, were included in the study, with 69.2% being males. Prenatal causes accounted for half of the cases, with genetic abnormalities (32.7%) and chromosomal abnormalities (7.7%) being prominent. Perinatal causes were the next most common (34.6%), including hypoxic-ischemic encephalopathy (26.7%). Postnatal causes were rare (3.8%). The overall etiological yield was 88.4%, with some cases remaining unidentified. Conclusions Prenatal causes, including genetic and chromosomal abnormalities, are common in GDD. The utilization of genetic testing enhances etiological yield. Hypoxic-ischemic encephalopathy remains a significant factor and highlights the importance of perinatal care in preventing developmental delays. Large multicentric studies are needed for a comprehensive database of etiological profiles.

The Clinical Profile of Children With Hepatitis A Infection: An Observational Hospital-Based Study.

INTRODUCTION:  Hepatitis A is a frequent form of hepatitis, especially in children. The changing epidemiology of the disease signifies the need for descriptive data concerning the clinical presentation and outcome of hepatitis A in children. The present study describes the clinical and biochemical profile of children with hepatitis A infection from a tertiary care center in the Aurangabad district of Maharashtra in Western India. METHODS:  One hundred patients between one and 18 years of age, presenting with symptoms/signs such as nausea, anorexia, vomiting, jaundice, abdominal pain, tender hepatomegaly, bleeding manifestations, or encephalopathy, were enrolled for the study. Serologically confirmed cases by detecting immunoglobulin M (IgM) antibodies against hepatitis A virus (HAV) were enrolled in the study. A detailed case proforma noted the clinical features and details such as age, gender, area, water supply, socioeconomic status, season, and biochemical parameters. RESULTS:  Most patients (45%) were among the age group of one to five years. Fever was reported in 96 (96%) patients, abdominal pain in 78 (78%) patients, dark-colored urine in 65 (65%) patients, vomiting in 47 (47%) patients, and anorexia in 63 (63%) patients. Icterus was found in 80 (80%) patients and hepatomegaly in 74 (74%) patients. In 61 (61%) patients, serum total bilirubin level on the first day was 1-4 mg/dL. Sixty-five (65%) patients were using tap water as the water source, and the maximum number of patients (43%) came in August. Most patients belonged to the class IV group (61%) as per the modified Kuppuswamy classification. CONCLUSIONS:  Most patients were 10 years or below, presenting predominantly with fever, abdominal pain, dark-colored urine, vomiting, and anorexia. Icterus and hepatomegaly were found in three-fourths or more of the patients. Around monsoon (June to September), there was the highest frequency of cases, and the socioeconomic status of most of the patients was within lower or upper-lower categories.

Spectrum of Common and Rare Small Molecule Inborn Errors of Metabolism Diagnosed in a Tertiary Care Center of Maharashtra, India.

Introduction Inborn errors of metabolism (IEM) form a large group of genetic diseases involving defects in genes coding for enzymes, receptors, and cofactors in the metabolic pathways of small and large molecules. The present study is the comprehensive data analysis of the tandem mass spectrometry (TMS) and urine metabolic pattern for the diagnosis of IEMs by gas chromatography and mass spectrometry (GC/MS) in samples received for high-risk IEM screening. Methods We conducted a retrospective analysis of children diagnosed with IEMs presenting at the genetic clinic of Mahatma Gandhi Missions (MGM) Medical College, Aurangabad. This article summarizes retrospective data of 40 pediatric cases over a three-year period, diagnosed with small molecule IEM based on the standard testing criteria. Results Out of 40, 17 patients (42.5%) were found to have organic acidemias, four (10%) had fatty acid oxidation defects, six (15%) had disorders of aminoacidopathies, seven (17.5%) had mitochondrial diseases, and three (7.5%) had urea cycle defects. One patient in each group (2.5% each) had carbohydrate metabolism defects, purine metabolic defects, and neurotransmitter metabolic defects. Conclusions This clinico-etiological profile study has thrown light on the clinical features and natural course of many common and rare IEMs, and it may provide clinicians with a deeper understanding of these conditions, allowing for improved early diagnosis and treatment of these diseases. Because of the high degree of consanguinity and marriages in the same community, common as well as many rare inherited metabolic diseases were diagnosed and novel genetic variants were identified.

Calcitonin receptor is required for T-antigen-induced prostate carcinogenesis.

Expression of calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancer (PC) and activation of CT-CTR axis in non- invasive PC cells induces an invasive phenotype. However, the role of CT-CTR axis in prostate carcinogenesis has not been investigated. We employed a transgenic mouse prostate cancer model that uses long probasin promoter to target the expression of T-antigen in the prostate gland (LPB-Tag) along with CTR knock-out mice (CTRKO) to address this question. We cross-bred LPB-Tag mice with CTRKO to obtain four groups of mice. Prostates of these mice were obtained at the age of 90 days, fixed, paraffin-embedded, and used either for the extraction of RNA or for immunofluorescence. Prostate RNAs from different groups were reverse transcribed and used either for transcription profiling or for qRT-PCR. As expected, prostates of mice with LPB-Tag genotype displayed well-grown tumors with histologic features such as loss of normal morphology and nuclear atypia. WT as well as CTRKO mice displayed normal prostate morphology. Interestingly, LPB-Tag-CTRKO prostates also displayed relatively normal morphology which was indistinguishable from the WT. Microarray analysis as well as qRT-PCR suggested that CTRKO genotype reversed T-antigen-induced silencing of RB and PTEN gene expression as well as T-antigen-induced expression of several enzymes associated with lipid metabolism/ cholesterol biosynthesis, several cancer-related and androgen-regulated genes. The results for the first time identify mechanisms associated CTR-induced prostate carcinogenesis, and raise an exciting possibility of using a potent CT antagonist to attenuate progression of prostate cancer.

The Effects of Matrigel® on the Survival and Differentiation of a Human Neural Progenitor Dissociated Sphere Culture.

We have previously developed an in vitro organotypic culture setting in order to investigate the performance of cellular substrates transplanted to the auditory nervous system. We have utilized this system to predict the efficacy of human neural progenitor cells (HNPCs) in transplantation to the auditory nerve to facilitate regeneration of sensory auditory nerve structures in vivo and in vitro. To optimize the growth and differentiation of HNPCs we have introduced an expansion of our in vitro system, exploring the impact of a growth factor-altered microenvironment. Here, we seeded HNPCs as a dissociated sphere culture on a hydrogel matrix coating (Matrigel®). We evaluated the performance of HNPCs by studying their survival, differentiation, and their axon-forming capacity. In identical culture conditions, we found that the overall survival rate of HNPCs on Matrigel coated surfaces was better than that on surfaces that were not coated with Matrigel. Furthermore, cells on Matrigel differentiated into neuronal cells to a far greater extent leading to strong synaptic marker signatures. Overall, our findings show that the present Matrigel matrix setting offers an experimental environment for the HNPCs to grow where these cells show novel and promising phenotypic characteristics suitable for further in vivo transplantation to the auditory nerve. Anat Rec, 303:441-450, 2020. © 2019 American Association for Anatomy.

Impact of recurring intermediate insulin-induced hypoglycemia on hypothalamic paraventricular corticotropin-releasing hormone, oxytocin, vasopressin and glucokinase gene profiles: role of type II glucocorticoid receptors.

Activation of central type II glucocorticoid receptors (GR) during neutral protamine Hagedorn insulin (NPH) administration exacerbates recurring hypoglycemia. The hypothalamic paraventricular nucleus (PVN) integrates metabolic sensory input, controls autonomic and neuroendocrine motor outflow, and is characterized by abundant GR expression. The present studies investigated the hypothesis that PVN GR mediate intensification of hypoglycemia by serial NPH dosing, and that PVN glucokinase (GCK) and glucoregulatory neuropeptide genes acclimate to this treatment paradigm through GR-dependent mechanisms. Groups of adult male rats were injected subcutaneously with one or four doses of NPH, on as many days, while controls received vehicle. Bilateral administration of the selective GR antagonist, CP-472555, into the PVN prior to the first three NPH injections prevented amplification of hypoglycemia in response to the final insulin dose, while intra-PVN delivery of the GR agonist, dexamethasone, to euglycemic rats did not modify ensuing NPH-induced hypoglycemia. Quantitative real-time RT-PCR analysis of microdissected PVN tissue revealed that GCK, corticotropin-releasing hormone (CRH), oxytocin (OT), and vasopressin (VP) mRNA levels were unchanged in response to acute NPH, and baseline gene profiles measured 24 h after antecedent injections were similar to vehicle controls. In contrast, serial dosing with NPH elevated CRH and GCK, diminished OT, but did not alter VP gene transcripts. Intracerebroventricular CP-472555 delivery in conjunction with antecedent NPH dosing prevented transcriptional habituation of GCK and OT genes, but did not modify CRH or VP mRNA profiles. The present data show that activation of PVN GR during antecedent intermediate insulin-induced hypoglycemia is required for exacerbation of recurring hypoglycemia, and receptor stimulation in the absence of hypoglycemia and/or its sequelae does not intensify the effects of subsequent NPH administration. The results also provide evidence for acclimation of PVN CRH, GCK, and OT gene profiles to serial NPH dosing, and demonstrate that GR may be involved in GCK and OT transcriptional adaptation to ongoing intermediate insulin-induced hypoglycemia.

Calcitonin induces stem cell-like phenotype in prostate cancer cells.

Stem cell-like-cancer cells are key drivers of tumor growth, metastasis, and relapse of cancer following remission. Prostate stem cell-like cancer cells isolated from human prostate cancer (PC) biopsies express CD44+/α2ß1 hi/CD133+ cell surface markers and can self-renew in vitro. Expression of calcitonin (CT) and its receptor (CTR) is frequently elevated in PCs and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We investigated whether CT-CTR autocrine axis induces stem cell-like phenotype in two PC cell lines. CT-CTR axis in these cell lines was activated by enforced expression of CTR. The cells were then examined for the changes in the expression of CD44 and CD133, collagen adherence, tumorigenic, metastatic and repopulating characteristics. The activation of CT-CTR axis led to a large increase in adherence to collagen and a remarkable increase of CD44 and CD133 in PC-3 and LNCaP cells. This was accompanied by a strong increase in tumorigenic, metastatic and repopulation properties of PC cells. However, the mutation of CTR-C PDZ-binding site in CTR almost abolished CTR-mediated increases in stem cell-like characteristics of PC cells. These results support an important role for CT-CTR axis in the progression of PC from localized cancer to an aggressive form, and a majority of proinvasive CTR actions may be mediated through its interaction with its partner protein at the PDZ-binding site. These results suggest that CT/CTR can serve as a valuable target to prevent the generation of stem-like PC cells.

Role of dorsal vagal motor nucleus orexin-receptor-1 in glycemic responses to acute versus repeated insulin administration.

The potent orexigenic neuropeptide, orexin-A (ORX-A), acts at multiple sites within the central neuroaxis to control autonomic responses to energy imbalance, including the dorsal vagal motor nucleus (DMV), where it regulates pancreatic efferent nerve firing. Recent evidence that recurrent insulin-induced hypoglycemia (RIIH) attenuates lateral hypothalamic ORX-A-ergic neuronal transcriptional activation and prepro-orexin gene expression suggests that this phenotype undergoes functional adaptation to repeated glucoprivation. We examined the hypothesis that RIIH-associated patterns of ORX-A neurotransmission and/or orexin-receptor-1 (OR-1) expression within the DMV may be correlated with exacerbated hypoglycemic and impaired pancreatic counterregulatory responses to repeated insulin administration. Male rats were pretreated by bilateral intra-DMV infusion of the OR-1 antagonist, SB-334867, or vehicle prior to s.c. injection of Humulin NPH (NPH), or diluent alone. Other animals were injected with one or four doses of NPH, on as many days, or diluent alone, and pretreated by bilateral intra-DMV administration of graded doses of ORX-A or vehicle on the final day of the study. Effects of acute versus repeated insulin administration on ORX-A and OR-1 protein levels in the microdissected dorsal vagal complex (DVC) were evaluated by radioimmunoassay and Western blot analyses, respectively. SB-334867 treatment prior to acute NPH administration decreased plasma glucose and suppressed peak glucagon secretion, whereas exogenous ORX-A administration prior to RIIH did not reverse amplified patterns of hypoglycemia. RIIH did not alter intra-DVC ORX-A tissue concentrations, but diminished OR-1 levels in that site. These results show that DMV OR-1 function is critical for optimal glucagon secretory responsiveness to acute hypoglycemia, and that RIIH-associated downregulation of receptor expression in that brain site may contribute to impaired restoration of euglycemia. The current data provide unique evidence that ORX-A acts via OR-1-dependent mechanisms within DMV to regulate glucagon counterregulatory function during hypoglycemia, and that decreased receptor-mediated signaling during RIIH may underlie characteristic intensification of hypoglycemia.

Type II glucocorticoid receptor involvement in habituated activation of lateral hypothalamic area orexin-A-immunopositive neurons during recurring insulin-induced hypoglycemia.

Neurons that synthesize the potent orexigenic neuropeptide, orexin-A (ORX-A) are confined to the lateral hypothalamic area (LHA) and adjacent structures, and project throughout the central neuroaxis to structures that govern central nervous system responses to energy imbalance. Insulin-induced hypoglycemia (IIH) upregulates prepro-orexin mRNA and Fos immunostaining of LHA ORX-A neurons. These neurons apparently become desensitized to this metabolic challenge, since both responses are diminished by recurrent insulin-induced hypoglycemia (RIIH). Recent studies implicate central type II glucocorticoid receptors (GR) in RIIH-associated glucose counterregulatory collapse and decline in Fos labeling of central metabolic loci, including the LHA. The present studies evaluated the role of GR in patterns of LHA ORX-A neuronal transcriptional activation during RIIH. Groups of adult male rats were injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, on as many days, or with diluent alone. Rats injected with four doses of insulin were pretreated by intracerebroventricular (icv) administration of the selective GR antagonist, CP-472555, or the vehicle, propylene glycol, prior to insulin administration on days 1-3. All animals were sacrificed by transcardial perfusion 2h after injections on day 4. Processing of LHA tissue sections for dual-immunoperoxidase staining of ORX-A- and Fos-immunoreactivity (-ir) showed that colabeling of ORX-A neurons for Fos was increased by a single injection of NPH, whereas this genomic response was diminished by RIIH. Icv administration of CP-472555 during antecedent hypoglycemia prevented RIIH-associated reductions in Fos expression by these neurons. Antagonist treatment of diluent-injected controls did not alter mean numbers of ORX-A- plus Fos-ir neurons. Total numbers of ORX-A-immunopositive neurons were not different among treatment groups. These data demonstrate that precedent central GR blockade prevents adaptation of LHA ORX-A neuronal reactivity to RIIH. These results provide unique pharmacological evidence that hypoglycemic hypercorticosteronemia diminishes activation of this neurotransmitter phenotype in this critical metabolic structure to subsequent hypoglycemia via central GR-dependent mechanisms.

Habituation of insulin-induced hypoglycemic transcription activation of lateral hypothalamic orexin-A-containing neurons to recurring exposure.

A CNS component of glucose counterregulatory collapse is supported by evidence for nonuniform genomic responsiveness of neurons in characterized central autonomic loci during recurring insulin-induced hypoglycemia (IIH). We have reported that exacerbated hypoglycemia and attenuated patterns of glucagon and epinephrine secretion in rats treated by daily sc injection of the intermediate-acting insulin formulation, Humulin NPH (NPH), are correlated with diminished immunodemonstrability of the AP-1 transcription factor, Fos, in several components of the central metabolic regulatory circuitry, including the lateral hypothalamic area (LHA). Neurons that synthesize the potent orexigenic peptide neurotransmitter, orexin-A, are restricted to the LHA and adjacent hypothalamic loci, and project throughout the central neuroaxis to structures that govern autonomic and behavioral motor output. Dual-label immunocytochemical and real-time RT-PCR techniques were utilized here to evaluate the functional status of this LHA phenotype during a single versus repetitive exposure to prolonged IIH. Tissue sections were collected at predetermined rostrocaudal levels of the LHA after acute or repeated NPH administration, and processed for nuclear Fos- and cytoplasmic orexin-A-immunoreactivity (-ir). Mean numbers of orexin-A-ir neurons were not different between treatment groups. Colabeling of these cells for Fos was increased relative to controls following a single injection of insulin, but numbers of Fos-ir-positive orexin-A neurons were significantly reduced after treatment with four versus one dose of insulin. Prepro-orexin mRNA levels in microdissected LHA tissue were upregulated during acute hypoglycemia, but were returned to control levels by repeated IIH. These data corroborate previous evidence that IIH is an activational stimulus for orexin-A-synthesizing neurons in the LHA, and further demonstrate that induction of cfos and prepro-orexin gene expression by acute hypoglycemia is attenuated by precedent exposure to hypoglycemia. The current results thus provide unique evidence for neurotransmitter-specific habituation of LHA neuronal sensitivity to IIH.

Effects of acute and chronic insulin-induced hypoglycemia on type II glucocorticoid receptor (GR) gene expression in characterized CNS metabolic loci.

The metabolic stressor, hypoglycemia, elicits integrated counterregulatory responses, including activation of the hypothalamic-pituitary-adrenal axis. Type II glucocorticoid receptors (GR) occur in multiple components of the central autonomic circuitry that regulates glucostasis, and antecedent GR stimulation is implicated in impaired glucagon and counterregulatory dysfunction during recurrent insulin-induced hypoglycemia (RIIH). To examine the hypothesis that this chronic stress may alter basal and/or hypoglycemic patterns of GR gene expression in a site-specific manner, real-time RT-PCR techniques were utilized to evaluate tissue GR mRNA levels in the microdissected lateral hypothalamic area (LHA) and paraventricular (PVH), dorsomedial (DMH), ventromedial (VMH), and arcuate (ARH) hypothalamic nuclei, before and after one or four injections, on as many days, of the intermediate-acting insulin formulation, Humulin NPH (NPH), while controls were treated with diluent alone. Rats injected with four doses of NPH were pretreated by intracerebroventricular (icv) administration of the selective nonsteroidal GR antagonist, CP-472555, or vehicle alone prior to insulin injections on days 1-3. The results show that acute hypoglycemia had no impact on GR mRNA levels in each structure evaluated. Basal GR gene expression was not altered by antecedent hypoglycemia, but tissue transcript levels were elevated in the DMH, PVH, VMH, and ARH during RIIH. Icv CP-472555 administration prior to antecedent hypoglycemia prevented RIIH-associated increases in GR mRNA in each of these sites. These data show that GR gene transcripts are increased in discrete CNS metabolic loci during RIIH, and that these local responses are attenuated by pharmacological blockade of central GR activation. The present studies demonstrate that hypoglycemic hypercorticosteronemia causes upregulated GR gene expression during RIIH, and implicate GR in mechanisms underlying this action.

BDNF increases survival and neuronal differentiation of human neural precursor cells cotransplanted with a nanofiber gel to the auditory nerve in a rat model of neuronal damage.

OBJECTIVES: To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation. METHODS: We transplanted HNPCs to the rat AN trunk by the internal auditory meatus (IAM). Furthermore, we studied if addition of BDNF affects survival and phenotypic differentiation of the grafted HNPCs. A bioactive nanofiber gel (PA gel), in selected groups mixed with BDNF, was applied close to the implanted cells. Before transplantation, all rats had been deafened by a round window niche application of ß-bungarotoxin. This neurotoxin causes a selective toxic destruction of the AN while keeping the hair cells intact. RESULTS: Overall, HNPCs survived well for up to six weeks in all groups. However, transplants receiving the BDNF-containing PA gel demonstrated significantly higher numbers of HNPCs and neuronal differentiation. At six weeks, a majority of the HNPCs had migrated into the brain stem and differentiated. Differentiated human cells as well as neurites were observed in the vicinity of the cochlear nucleus. CONCLUSION: Our results indicate that human neural precursor cells (HNPC) integration with host tissue benefits from additional brain derived neurotrophic factor (BDNF) treatment and that these cells appear to be good candidates for further regenerative studies on the auditory nerve (AN).

Brain stem slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture.

Conditioned medium (CM), made by collecting medium after a few days in cell culture and then re-using it to further stimulate other cells, is a known experimental concept since the 1950s. Our group has explored this technique to stimulate the performance of cells in culture in general, and to evaluate stem- and progenitor cell aptitude for auditory nerve repair enhancement in particular. As compared to other mediums, all primary endpoints in our published experimental settings have weighed in favor of conditioned culture medium, where we have shown that conditioned culture medium has a stimulatory effect on cell survival. In order to explore the reasons for this improved survival we set out to analyze the conditioned culture medium. We utilized ELISA kits to investigate whether brain stem (BS) slice CM contains any significant amounts of brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF). We further looked for a donor cell with progenitor characteristics that would be receptive to BDNF and GDNF. We chose the well-documented boundary cap (BC) progenitor cells to be tested in our in vitro co-culture setting together with cochlear nucleus (CN) of the BS. The results show that BS CM contains BDNF and GDNF and that survival of BC cells, as well as BC cell differentiation into neurons, were enhanced when BS CM were used. Altogether, we conclude that BC cells transplanted into a BDNF and GDNF rich environment could be suitable for treatment of a traumatized or degenerated auditory nerve.

Site-specific habituation of insulin-induced hypoglycemic induction of fos immunoreactivity in glucocorticoid receptor: immunopositive neurons in the male rat brain.

Current studies show that type II glucocorticoid receptor (GR) stimulation during recurring insulin-induced hypoglycemia (RIIH) results in diminished hypoglycemic activation of neurons in discrete CNS metabolic structures, namely the lateral hypothalamic area (LHA), hypothalamic paraventricular (PVH) and dorsomedial (DMH) nuclei, and nucleus of the solitary tract (NTS). The present work utilized immunofluorescence histochemistry to evaluate the reactivity of GR-expressing neurons in characterized hypothalamic, thalamic, and hindbrain metabolic structures to glucoprivation, and to determine if antecedent hypoglycemic stimulation of central GR decreases Fos protein expression by these neurons. Groups of adult male rats were injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, on as many days, while controls received diluent only. Rats injected with four doses of insulin were pretreated by intracerebroventricular administration of the selective GR antagonist, CP-475222, or vehicle alone prior to insulin doses 1-3. All animals were sacrificed by trancardial perfusion 2 h after injections on day four of the study. Mean numbers of GR-immunoreactive (-ir) neurons did not differ between groups injected with diluent versus one dose of insulin in each structure evaluated, but were significantly elevated above baseline on the fourth day of RIIH in the LHA and DMH, but not the PVH, VMH, ARC, thalamic paraventricular (PVT), or NTS. Counts of GR-ir-positive neurons in each site were similar between groups treated with CP-475222 or vehicle icv during RIIH. While mean numbers of GR-plus Fos-ir neurons in the PVH, DMH, LHA, and NTS, but not the PVT were significantly elevated after one dose of NPH, this increase was abolished in each site by RIIH. Pharmacological antagonism of central GR during antecedent hypoglycemia prevented RIIH-associated habituation of Fos colabeling of GR-expressing neurons in the PVH, DMH, and LHA. These data show that RIIH increases nuclear immunolabeling for GR in discrete CNS metabolic structures, evidence that recurring metabolic stress may amplify receptor-mediated genomic regulatory function in local neurons. The results also demonstrate that GR-containing neurons in the LHA, DMH, PVH, and NTS react to hypoglycemia by induction of the Fos stimulus-transcription cascade, and that precedent stimulation of central GR is critical for RIIH-associated habituation of this functional response in the former three sites.