Immunohistochemical examination of the INK4 and Cip inhibitors in the rat neonatal cerebellum: cellular localization and the impact of protein calorie malnutrition.

Expression of the cyclin-dependent kinase inhibitors (CKIs) has been linked to the inhibition of cellular proliferation and the induction of differentiation. Based on structure function analysis, two distinct families of CDKIs, the INK4 and the Cip/Kip family have been identified. The INK4 family member p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in normal development of the CNS and cerebellum. Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D1 in orchestrating growth factor-induced cellular proliferation. The neonatal rat cerebellum undergoes proliferative growth and differentiation, localized to distinct topographical regions and cell types. The cell type and the temporal profile of CKI expression during postnatal cerebellar development had not been described. The current studies determined the specific cerebellar cell types in which the CKIs were expressed during post natal development by co-staining for cell-type specific markers. p16(Ink4a) and p27(Kip1) immunostaining was identified in both neurons and glial cells, increasing progressively between postnatal days 6 to 13 into adulthood. By contrast, neuronal and glial cell p21(Cip1) staining was prominent at days 6-11 and decreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal granular layers. Dual immunostaining demonstrated cyclin D1 within cells expressing CKI (p16(Ink4a), p21(Cip1),p27(Kip1)). Cerebellar cellular growth arrest, induced by protein-calorie malnutrition, inhibited cyclin D1 protein levels without affecting CKI immunostaining suggesting CKI do not mediate the developmental arrest. These results demonstrate that the CKIs are induced by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo.

Regulation of cyclin dependent kinase inhibitor proteins during neonatal cerebella development.

The cyclin dependent kinase holoenzymes (CDKs), composed of catalytic (cdk) and regulatory (cyclin) subunits, promote cellular proliferation and are inhibited by cyclin dependent kinase inhibitor proteins (CDKIs). The CDKIs include the Ink4 family (p15Ink4b, p16Ink4a, p18Ink4c, p19Ink4d) and the KIP family (p21Cip1 and p27Kip1). The sustained induction of p21 and p18 during myogenesis implicates these CDKI in maintaining cellular differentiation. Herein we examined the CDK (cyclin D1, cdk5) and CDKI expression profiles during the first 24 days of postnatal rat cerebella development. Cdk5 abundance increased and cyclin D1 decreased from day 9 through to adulthood. The CDKIs increased transiently during differentiation. p27 increased 20-fold between days 4 and 24, whereas p21 rose twofold between 6 to 11 days. p19, p18 and p16 increased approximately two- to threefold, falling to low levels in the adult. Immunostaining of cyclin D1 was localized in the external granular cells, whereas p27, was found primarily in the Purkinje cells. The period of maximal differentiation between days 9 to 13 was associated with a change in p21 and p16 staining from the external granular and Purkinje cells to a primarily Purkinje cell distribution. Protein-calorie malnutrition, which was previously shown to arrest rat cerebella development, reduced cyclin D1 kinase activity and p27 levels. However, p16 and p21 levels were unchanged. We conclude that the CDKIs are induced with distinct kinetics in specific cell types and respond differentially to growth factors during cerebella development, suggesting discrete roles for these proteins in normal cerebella development.

Alteration in IGF-I binding in the cerebral cortex and cerebellum of neonatal rats during protein-calorie malnutrition.

Neonatal brain development in the rat is adversely affected by malnutrition. Alterations in tissue binding of IGF-I in the malnourished brain were tested in rat pups from mothers who were fed a 20% protein diet (C) or a 4% protein diet (M) starting from day 21 of gestation and continued throughout suckling. IGF-I binding in both cortex and cerebellum decreased progressively in C and M groups from day 6 to day 13. At day 9, 11, and 13, the binding was significantly greater (p < 0.02) in M compared to C groups. To investigate whether these changes might be related to the alteration in receptor activity, membranes were incubated with 125I-IGF in the presence of excess insulin with or without unlabeled IGF-I. In the absence of insulin, specific IGF-I binding in the M group was increased by 41.8 +/- 13.8% (mean +/- SEM p < 0.05) relative to C group. Insulin produced a consistent but incomplete inhibition of binding in both C and M, of 75% and 67% respectively. In addition, the specific IGF-I binding in the presence of insulin was increased in M group by 70.2 +/- 9.4% relative to C, p < 0.05. To characterize the nature of this binding, cerebral cortical membranes, from both groups, incubated with 125I-IGF-I were cross-linked, and electrophoresed on 6% and 10% SDS-PAGE gels under reducing conditions. Autoradiography of the 6% gel showed two specific bands at 115 kD and 240 kD, consistent with monomeric and dimeric forms of the IGF-I receptor, which were inhibited by excess insulin. In contrast, a 10% gel showed an additional band at 35 kD (IGF-binding protein) that was not inhibited by insulin. In both gels, membrane preparations from the M group showed a heightened intensity of the bands relative to C. The increase in binding protein relative to the receptor suggests a disequilibrium that may limit the availability of exogenous IGF-I to the tissues.

Pathophysiological mechanisms in immune inner ear disease.

Immune inner ear disease is a somewhat controversial entity which describes cochleovestibular dysfunction that is related to immune-mediated mechanisms. The diagnosis of this disease is based on clinical presentation and response to various treatment protocols. Unfortunately, the presentation is variable and the treatment empirical, and this has caused much confusion in the diagnosis and management of the condition. To elucidate the variable nature of the disease, it is important to understand that more than one mechanism of immune injury may be involved. This paper attempts to classify clinical and experimental cases of immune inner ear disease with regard to the Gell and Coombs classification scheme of immune-mediated injury. By understanding the different pathophysiological mechanisms involved, the clinician should be better able to diagnose and manage this difficult problem in a directed fashion. The ramifications of the proposed classification system on the diagnosis, treatment and future research of immune inner ear disease are discussed.

Reduced cyclin D1 expression in the cerebella of nutritionally deprived rats correlates with developmental delay and decreased cellular DNA synthesis.

Nutritional deprivation in the early postnatal period severely inhibits cerebellar growth and development, which is related in part to reduced levels of growth factors. Cyclin D1 encodes a growth factor-inducible regulatory subunit of a serine/thereonine kinase that is capable of phosphorylating the tumor suppressor pRB, thereby allowing normal progression through the G1 phase of the cell-cycle. Because the abundance of cyclin D1 is rate limiting in this progression, we examined the regulation of cyclin D1 expression in vivo, using a model of nutritional deprivation. Cyclin D1 expression in cerebella of fed control rats was detected in the external granular layer and was associated with cellular proliferation within this layer. Nutritional deprivation of rats reduced cerebellar weight, as well as the thickness of the molecular layer that largely consists of cells migrating from the external granular layer. Refeeding partially restored cerebellar weight, molecular layer thickness and increased external granular layer cyclin D1 immunostaining. Since nutritional deprivation is accompanied by lower levels of circulating insulin-like growth factor-I (IGF-I), we determined whether IGF-I directly stimulated the cyclin D1 promoter. The human cyclin D1 promoter linked to the luciferase reporter gene was stably integrated into PC12 cells. IGF-I stimulated cyclin D1 promoter activity 4- to 6-fold at 6 hours (h). These findings are consistent with the notion that nutritional deprivation may affect proliferative growth by altering expression of cyclin D1 in the germinal cell layer and that regulation of cyclin D1 expression by growth factors may contribute to normal neonatal cerebellar development. The reduction in cyclin D1 expression as cells differentiate in the cerebellum is consistent with a potential role for cyclin D1 in this process.

Nutritional insult and recovery in the neonatal rat cerebellum: insulin-like growth factors (IGFs) and their binding proteins (IGFBPs).

Alterations in growth caused by neonatal malnutrition may be mediated in part by changes in insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression. Since the neonatal rat cerebellum undergoes a transient, proliferative growth phase in the first two weeks of life, this structure was used to determine whether alterations in circulating and tissue IGFs and IGFBPs may mediate effects of impaired nutrition on the developing central nervous system. Gravid rats were placed on a 4% (protein-calorie deprived, D) or 20% (control, C) protein diets one day prior to delivery and allowed to nurse their pups postpartum. Pups nursing from D mothers received a limited volume of milk and were calorically deprived. Some litters of D pups were foster fed by C mothers from day 8 to day 13 to constitute a recovery group (R). Cerebellar weight, protein, and DNA content in D pups were less than C, p < 0.001. In R pups, DNA and protein returned to C levels by day 13. Between days 6 and 13, serum IGF-I levels rose from 158 +/- 18 to 210 +/- 18 ng/ml in C but remained low in D (47 +/- 6 ng/ml and 25 +/- 3 ng/ml), respectively. In R pups, serum IGF-I partially recovered during this time, and increased from 49 +/- 5 to 110 +/- 7 ng/ml. In cerebellar extracts, IGF-I levels in both C and D were lower at 13 days than at 6 days, p < 0.05 and p < 0.005, respectively. IGF-I levels in C were similar at day 9 and day 11 and were consistently higher than D (11.84 +/- 0.83 vs 8.56 +/- 0.92 ng/g, p < 0.02 C vs D). In R, IGF-I was reduced on day 11, but was similar to C on day 13. Serum IGF-II in D was lower than C, p < 0.01, and did not increase in the R group. Cerebellar IGF-II was virtually undetectable in either group. Immunoprecipitation and ligand blotting studies of serum demonstrated that circulating levels of 32-34 K IGFBPs were increased 3-4 fold in D vs C, reflecting high levels of IGFBP-1 and/or -2, while levels of 24 K IGFBP-4 were lower in D vs C. By contrast, immunoprecipitation and ligand blotting of cerebellar extracts detected IGFBP-2 and -4, but did not detect IGFBP-1. Further, tissue levels of IGFBP-2 were not increased in D vs C, and levels of IGFBP-4 also were not markedly affected by nutritional deprivation. These results suggest that alterations in tissue content and the availability of IGF-I only modestly contributed to the effects of impaired nutrition in the developing central nervous system.

Adaptation of the diphenylamine (DPA) assay to a 96-well plate tissue culture format and comparison with the MTT assay.

A simple spectrophotometric method for measuring DNA in proliferating cells is described. The method is an adaptation of the widely used diphenylamine (DPA) reaction to examine DNA in cells grown in a 96-well plate. This assay was capable of detecting as little as 10 ng DNA and could be used to measure DNA in stored as well as viable tissue samples. The DPA assay paralleled the MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay of mitochondrial reductase in A549 cells, a human lung cancer cell line; in EMT6 cells, a mouse breast cancer cell line; and in a primary cell culture of neonatal rat astrocytes. Over several days of proliferative growth in tissue culture, the ratio of MTT to DPA remained constant. Since the DPA assay and MTT assay measured different parameters of the same cells, they could be employed as complementary spectrophotometric assessments of cell growth on a 96-well plates using the same automated enzyme-linked immunosorbent assay plate reader.

Proliferative growth of neonatal cerebellar cells in culture: regulation by male and by maternal serum in late gestation.

Neonatal cerebellar cells were utilized as a model system to examine the effect of 20 day pregnant rat serum on proliferative growth in the CNS. Cells were prepared by mechanical dissociation and cultured as mixed cells or populations enriched in astrocytes or oligodendrocytes. Cell proliferation was estimated by measurement of DNA, protein, and/or mitochondrial reductase activity (MTT). When mixed cells were incubated with 10% male rat serum, both total DNA and protein content increased after 6 days of culture. By contrast, neither of these parameters were altered in cultures incubated with 10% pregnant serum. When cells were incubated with either male or pregnant sera, changes in MTT activity paralleled changes in protein content. Graded concentrations of pregnant serum (5-20%) added to mixed cell cultures produced consistently lower MTT values when compared with identical concentrations of male serum. In addition, MTT activity was diminished in both astrocytes and oligodendrocytes incubated with graded concentrations of pregnant sera when compared with similar concentrations of non-pregnant sera. When potential effects of these different sera on the cell cycle were examined, an increase in the number of cells in the S and G2/M phase was similar, and DNA doubling began to increase at 96 hrs in the presence of either male or 20 day pregnant sera. Thus the inhibition of cell growth by pregnant serum was not likely a result of either cytotoxicity or a delay of entry of cells into the cell cycle. To examine whether this inhibition of cell growth may reflect the effect of pregnant serum on endogenous growth factor production, we tested the production of IGF-II by cerebellar cells. production of an endogenous source of IGF-II was apparent using an RNAse protection assay and was noted using Slot Blot analysis of mRNA extracted at sequential times during cell incubation. Mixed cell cultures also secreted immunologically defined IGF-II. These observations are consistent with the previous demonstration that the fraction of pregnant serum which bound IGF-II also inhibited cell growth. The inhibitory effect of pregnant serum was diminished by preincubating aliquots of sera with graded concentrations of IGF-I prior to adding sera to tissue culture medium. Pregnant serum inhibition was also diminished by prolonging incubation times beyond 6 days. The blunting of pregnant serum inhibition may have been consequent to either a continuing production of endogenous growth factors or to the potential emergence of resistant cells due to prolonged tissue culture incubation.(ABSTRACT TRUNCATED AT 400 WORDS)

Sodium fluoride therapy.

As a medical treatment for otospongiosis-otosclerosis, sodium fluoride influences the underlying bony changes in the labyrinth so as to arrest or prevent the onset of hearing loss. Sodium fluoride is an enzyme inhibitor and reduces osteoclastic bone resorption and, if the dosage is more than 60 mg a day, may rebuild pseudohaversian bone.