Cavernous nerve injury elicits GAP-43 mRNA expression but not regeneration of injured pelvic ganglion neurons

Recovery of erectile dysfunction after cavernous nerve injury takes a long period. To elucidate this mechanism, unilateral cavernous nerve of male rat was cut, and the expression level of a nerve regeneration marker, the growth associated protein-43 (GAP-43) mRNA was evaluated by in situ hybridization and RT-PCR. While GAP-43 mRNA expression was transiently increased in the injured neurons of the major pelvic ganglion (MPG) at 7 days after nerve injury, continuous increase of GAP-43 mRNA was observed in the contralateral MPG from 7 days to 6 months after the nerve injury. Histochemical double-labeling studies for either neuronal NOS (nNOS) or tyrosine hydroxylase (TH) and the GAP-43 mRNA expression demonstrated that in injured MPG the transient up-regulation of GAP-43 mRNA was mainly seen in nNOS negative and/or TH positive neurons, suggesting non-parasympathetic post-ganglionic neurons, and also demonstrated that in contralateral MPG GAP-43 mRNA positive neurons were gradually increased in nNOS positive but TH negative neurons, suggesting parasympathetic post-ganglionic neurons. When a retrograde tracer Fluorogold (FG) was injected into the penile crus 7 days before histological experiments, FG-positive neurons were, if any, hardly seen in nNOS-positive neurons of the injured MPG for at least 6 months, whereas numerous FG-positive cells were seen in nNOS-positive neurons of the contralateral MPG. These results suggest that post-ganglionic projecting neurons of the intact side, which express increased GAP-43 mRNA, would be most likely to contribute to the recovery of the erectile function after unilateral cavernous nerve injury possibly by a plastic change such as nerve sprouting.     

Cell type- and region-specific expression of protein kinase C-substrate mRNAs in the cerebellum of the macaque monkey

We performed nonradioactive in situ hybridization histochemistry in the monkey cerebellum to investigate the localization of protein kinase C-substrate (growth-associated protein-43 [GAP-43], myristoylated alanine-rich C-kinase substrate [MARCKS], and neurogranin) mRNAs. Hybridization signals for GAP-43 mRNA were observed in the molecular and granule cell layers of both infant and adult cerebellar cortices. Signals for MARCKS mRNA were observed in the molecular, Purkinje cell, and granule cell layers of both infant and adult cortices. Moreover, both GAP-43 and MARCKS mRNAs were expressed in the external granule cell layer of the infant cortex. In the adult cerebellar vermis, signals for both GAP-43 and MARCKS mRNAs were more intense in lobules I, IX, and X than in the remaining lobules. In the adult hemisphere, both mRNAs were more intense in the flocculus and the dorsal paraflocculus than in other lobules. Such lobule-specific expressions were not prominent in the infant cerebellar cortex. Signals for neurogranin, a postsynaptic substrate for protein kinase C, were weak or not detectable in any regions of either the infant or adult cerebellar cortex. The prominent signals for MARCKS mRNA were observed in the deep cerebellar nuclei, but signals for both GAP-43 and neurogranin mRNAs were weak or not detectable. The prominent signals for both GAP-43 and MARCKS mRNAs were observed in the inferior olive, but signals for neurogranin were weak or not detectable. The cell type- and region-specific expression of GAP-43 and MARCKS mRNAs in the cerebellum may be related to functional specialization regarding plasticity in each type of cell and each region of the cerebellum.     

A disease-specific psychosocial questionnaire for Parkinson’s disease caregivers

Abstract. Objective: To evaluate the Belastungsfragebogen Parkinson Angehörigen—kurzversion (BELA-A-k), a questionnaire for measuring psychosocial problems and need for help in Parkinsons disease (PD) caregivers. Methods: The Belastungsfragebogen Parkinson Angehörigen—kurzversion was translated into Dutch. It consists of 15 items with a Bothered by (Bb) and a Need for Help (NfH) score. The BELA-A-k was tested for cultural differences, relevance and feasibility in a pilot (n = 10). We determined the psychometric properties in a validation study (n = 50) and compared the BELA-A-k with the Sickness Impact Profile, the COOP/WONCA Functional Health Assessment Charts and the Loneliness Questionnaire (de Jong-Gierveld). All questionnaires were administered in person at home, in a prescribed order. Results: The BELA-A-k was completed by 60 PD-caregivers. The internal-consistency reliability coefficients for the total Bothered by (0.90) and Need for Help (0.92) scales were excellent. The internal consistency of the subscales exceeded the 0.70 standard except for the Bothered by and Need for Help Social functioning scale (Bb = 0.62; NfH = 0.65) and the Partner-bonding/Family scale (NfH = 0.69). Almost all BELA-A-k subscales correlated highly (P < 0.001) with the corresponding scales of the standard quality of life indices. Conclusion: The BELA-A-k is a relevant, reliable and valid measure for assessing psychosocial problems and need for help of PDcaregivers.     

Northern blot and in situ hybridization analyses for the neurogranin mRNA in the developing monkey cerebral cortex

Neurogranin is a postsynaptic substrate for protein kinase C, and its expression is related to dendritic spine development and postsynaptic plasticity. Using both Northern blot analysis and in situ hybridization techniques, we investigated the developmental changes of neurogranin expression in the monkey cerebral cortex. In each of four neocortical areas examined, i.e., the prefrontal area (area FD of von Bonin and Bailey), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern blot analysis showed that the amount of neurogranin mRNA was low during the prenatal and perinatal periods until postnatal day 8. It increased during postnatal development and reached its peak value at postnatal day 70 (in area OC) or postnatal month 6 (in area FD, TE, and PB). After that, the amount of neurogranin mRNA in the cerebral neocortex decreased gradually until postnatal years 2-3. The in situ hybridization experiments also showed a transient increase of neurogranin mRNA in the neocortex during postnatal day 70 to postnatal month 6. The transient increase was prominent in layers II and III of areas FD and TE; deep in layer III of area PB; and in layers II, III, and IV of area OC. In the hippocampus, in contrast to the results in the neocortex, the expression of neurogranin mRNA was decreased almost continuously during the postnatal period. The transiently increased expression of neurogranin in the postnatal neocortex may be a molecular basis for the postsynaptic modification of afferent inputs possibly from subcortical structures.     

Glutamate release from astrocytes is stimulated via the appearance of exocytosis during cyclic AMP-induced morphologic changes

Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP-treated and non-treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP-treated astrocytes after stimulation with ATP by high-performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1-43 imaging, appeared in cyclic AMP-treated astrocytes in a punctiform fashion, but not in non-treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP-induced morphologic changes, and in particular, a v-SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP-treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP-induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP-treated astrocytes in response to ATP released from neighboring neurons and astrocytes.     

Relationship of calpain-mediated proteolysis to the expression of axonal and synaptic plasticity markers following traumatic brain injury in mice

The role of neuronal plasticity and repair on the final functional outcome following traumatic brain injury (TBI) remains poorly understood. Moreover, the relationship of the magnitude of post-traumatic secondary injury and neurodegeneration to the potential for neuronal repair has not been explored. To address these questions, we employed Western immunoblotting techniques to examine how injury severity affects the spatial and temporal expression of markers of axonal growth (growth-associated protein GAP-43) and synaptogenesis (pre-synaptic vesicular protein synaptophysin) following either moderate (0.5 mm, 3.5 M/s) or severe (1.0 mm, 3.5 M/s) lateral controlled cortical impact traumatic brain injury (CCI-TBI) in young adult male CF-1 mice. Moderate CCI increased GAP-43 levels at 24 and 48 h post-insult in the ipsilateral hippocampus relative to sham, non-injured animals. This increase in axonal plasticity occurred prior to maximal hippocampal neurodegeneration, as revealed by de Olmos silver staining, at 72 h. However, moderate CCI-TBI did not elevate GAP-43 expression in the ipsilateral cortex where neurodegeneration was extensive by 6 h post-TBI. In contrast to moderate injury, severe CCI-TBI failed to increase hippocampal GAP-43 levels and instead resulted in depressed GAP-43 expression in the ipsilateral hippocampus and cortex at 48 h post-insult. In regards to injury-induced changes in synaptogenesis, we found that moderate CCI-TBI elevated synaptophysin levels in the ipsilateral hippocampus at 24, 48, 72 h and 21 days, but this effect was not present after severe injury. Together, these data highlights the adult brain's ability for axonal and synaptic plasticity following a focal cortical injury, but that severe injuries may diminish these endogenous repair mechanisms. The differential effects of moderate versus severe TBI on the post-traumatic plasticity response may be related to the calpain-mediated proteolytic activity occurring after a severe injury preventing increased expression of proteins required for plasticity. Supporting this hypothesis is the fact that GAP-43 is a substrate for calpain along with our data demonstrating that calpain-mediated degradation of the cytoskeletal protein, alpha-spectrin, is approximately 10 times greater in ipsilateral hippocampal tissue following severe compared to moderate CCI-TBI. Thus, TBI severity has a differential effect on the injury-induced neurorestorative response with calpain activation being one putative factor contributing to neuroregenerative failure following severe CCI-TBI. If true, then calpain inhibition may lead to both neuroprotective effects and an enhancement of neuronal plasticity/repair mechanisms post-TBI.     

Chlorpromazine reduces the intercellular communication via gap junctions in mammalian cells

In the work presented herein, we evaluated the effect of chlorpromazine (CPZ) on gap junctions expressed by two mammalian cell types; Gn-11 cells (cell line derived from mouse LHRH neurons) and rat cortical astrocytes maintained in culture. We also attempted to elucidate possible mechanisms of action of CPZ effects on gap junctions. CPZ, in concentrations comparable with doses used to treat human diseases, was found to reduce the intercellular communication via gap junctions as evaluated with measurements of dye coupling (Lucifer yellow). In both cell types, maximal inhibition of functional gap junctions was reached within about 1 h of treatment with CPZ, an recovery was almost complete at about 5 h after CPZ wash out. In both cell types, CPZ treatment increased the phosphorylation state of connexin43 (Cx43), a gap junction protein subunit. Moreover, CPZ reduced the reactivity of Cx43 (immunofluorescence) at cell interfaces and concomitantly increased its reactivity in intracellular vesicles, suggesting an increased retrieval from and/or reduced insertion into the plasma membrane. CPZ also caused cellular retraction reducing cell-cell contacts in a reversible manner. The reduction in contact area might destabilize existing gap junctions and abrogate formation of new ones. Moreover, the CPZ-induced reduction in gap junctional communication may depend on the connexins (Cxs) forming the junctions. If Cx43 were the only connexin expressed, MAPK-dependent phosphorylation of this connexin would induce closure of gap junction channels.     

Schwann cells transplantation promoted and the repair of brain stem injury in rats

Objective To explore the possibility of Schwann cells transplantation to promote the repair of injured brain stem reticular structure in rats. Methods Schwann cells originated from sciatic nerves of 1 to 2-day-old rats were expanded and labelled by BrdU in vitro, transplanted into rat brain stem reticular structure that was pre-injured by electric needle stimulus, lmmunohistochemistry and myelin-staining were used to investigate the expression of BrdU, GAP-43 and new myelination respectively. Results BrdU positive cells could be identified for up to 8 months and their number increased by about 23%, which mainly migrated toward injured ipsilateral cortex. The GAP-43 expression reached its peak in 1 month after transplantation and was significantly higher than that in the control group. New myelination could be seen in destructed brain stem areas. Conelusion The transplantation of Schwann cells can promote the restoration of injured brain stem reticular structure.     

子宫平滑肌激活蛋白-1及间隙连接蛋白43在妊娠期的表达与分娩发动和早产的关系

目的　探讨激活蛋白-1(Activator protein-1,AP-1)、间隙连接蛋白43(connexin43,Cx43)在足月产和早产子宫平滑肌中的表达情况及其相关性. 方法　应用免疫组化法检测15例足月未临产、15例足月临产、10例早产临产产妇的子宫平滑肌中AP-1的两个亚单位c-Jun、c-Fos蛋白及Cx43的表达情况. 结果 (1)Cx43免疫组化积分在早产临产组(4.204±0.42)及足月临产组(4.33±0.51)表达水平显著高于足月未临产组(3.15±0.41)(P<0.01).(2)c-Jun蛋白在早产临产组、足月临产组、足月未临产组标记指数分别为(52.34±4.18)%、(45.25±5.24)%、(34.14±4.26)%,三组两两比较差异均有统计学意义(P<0.01).(3)c-Fos蛋白在早产临产组、足月临产组、足月未临产组标记指数分别为(53.48±4.36)%、(43.32±6.21)%、(31.29±3.34)%,三组两两比较差异均有统计学意义(P<0.01).(4)妊娠子宫平滑肌中Cx43表达与c-Jun、c-Fos蛋白表达呈正相关关系,(r=0.65、0.63,P<0.01). 结论 Cx43在分娩发动中发挥着重要的作用.妊娠子宫平滑肌中Cx43的表达水平与AP-1表达有一定的相关性.