Rosemary C. Borke

Brain stem afferents of hypoglossal neurons in the rat

The origin of afferent connections of the hypoglossal nucleus in rats was investigated using horseradish peroxidase (HRP) as a retrograde tracer. Pressure injections (0.15-0.17 mu1) of 15% HRP were introduced into the rostral, middle and caudal portions of the nucleus. Projections to the hypoglossal nucleus originated from 3 regions of the brainstem: the reticular formation, the spinal V complex and the nucleus of the solitary tract. Bilateral projections with ipsilateral predominance came from the lateral reticular formation: the dorsal aspect of the nucleus reticularis parvocellularis and its caudal continuation, the nucleus reticularis dorsalis. Fewer projections emerged from two nuclei of the medial reticular formation. The dorsal part of the nucleus reticularis ventralis at the spinomedullary junction contributed bilateral with mainly contralateral input to hypoglossal neurons. A few labeled neurons were situated bilaterally in the nucleus reticularis gigantocellularis of the rostral medulla. The input from the spinal V complex originated from the dorsal aspect along most of its length but particularly from the pars interpolaris and oralis subdivisions. Labeled neurons were located primarily in the posterior portion of the nucleus of the solitary tract. Projections from the spinal V complex and the solitary nucleus exhibited ipsilateral predominance. These results suggest that somatic and visceral centers of the rat brainstem play an important role in the control of the activity of hypoglossal motoneurons.

New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb

Ensheathing cells exclusively enfold olfactory axons. The ability of olfactory axons to reinnervate the adult mammalian olfactory bulb throughout the lifetime of an organism is believed to result from the presence of this unique glial cell in the olfactory system. This theory has been substantiated by research demonstrating the ability of transplanted ensheathing cells to promote axonal regrowth in areas of the central nervous system that are normally nonpermissive. A simple method for purifying ensheathing cells resulting in a large yield of cells is therefore invaluable for transplantation studies. We have developed such a method based on the differing rates of attachment of the various harvested cell types. The greatest percentage of cells (70.4%) that attached during the first step of the separation was determined to be fibroblasts. The remainder of the cells were classified as astrocytes (20.8%) and ensheathing cells (6.8%). The percentage of attached astrocytes (67.6%) was greatly increased during the second purification step while the percentage of fibroblasts decreased greatly (27.9%) and the percentage of ensheathing cells (5.3%) slightly decreased. In the final cultures, 93.2 % of the attached cells were ensheathing cells, while astrocytes (5.9%) and fibroblasts (1.4%) were only minor components. This simple, inexpensive method of purifying ensheathing cells will facilitate their use in central nervous system regeneration research. GLIA 34:81–87, 2001.

810 nm Wavelength light: An effective therapy for transected or contused rat spinal cord

Light therapy has biomodulatory effects on central and peripheral nervous tissue. Spinal cord injury (SCI) is a severe central nervous system trauma with no effective restorative therapies. The effectiveness of light therapy on SCI caused by different types of trauma was determined.

Experimental superficial siderosis of the central nervous system. I. Morphological observations.

Autologous washed red blood cells were injected weekly over a period of three to six months into the cisterna magna of adult New Zealand white rabbits. After three months, the surface of the brain stem, cerebellum, and piriform cortex showed a distinct brown color, and staining of the gross specimens for iron produced an intense blue color which extended for a distance of 1–2 mm into the brain parenchyma. Enhanced iron stains of vibratome sections revealed the accumulation of reaction product in microglia and Bergmann glia of the cerebellar cortex, and in microglia and astrocytes of the piriform cortex. Ferritin immunocytochemistry revealed reaction product in cerebellar microglia and Bergmann glia which strongly resembled that obtained by the enhanced iron stain. In the piriform cortex, only microglia were reactive with anti-ferritin. Electron microscopy confirmed the accumulation of electron-dense ferritin granules only in the cytoplasm of microglia. Bergmann glia in the cerebellum and astrocytic processes in the piriform cortex were replete with intermediate filaments and contained an excess of glycogen. After six months, small granules of hemosiderin began to appear in cerebellar and piriform cortices. The observations support that the sequence of conversion of hemoglobin to ferritin and hemosiderin occurs in brain as in other organs.

Abnormal expression of the G-protein-activated inwardly rectifying potassium channel 2 (GIRK2) in hippocampus, frontal cortex, and substantia nigra of Ts65Dn mouse: a model of Down syndrome.

Ts65Dn, a mouse model of Down syndrome (DS), demonstrates abnormal hippocampal synaptic plasticity and behavioral abnormalities related to spatial learning and memory. The molecular mechanisms leading to these impairments have not been identified. In this study, we focused on the G‐protein‐activated inwardly rectifying potassium channel 2 (GIRK2) gene that is highly expressed in the hippocampus region. We studied the expression pattern of GIRK subunits in Ts65Dn and found that GIRK2 was overexpressed in all analyzed Ts65Dn brain regions. Interestingly, elevated levels of GIRK2 protein in the Ts65Dn hippocampus and frontal cortex correlated with elevated levels of GIRK1 protein. This suggests that heteromeric GIRK1‐GIRK2 channels are overexpressed in Ts65Dn hippocampus and frontal cortex, which could impair excitatory input and modulate spike frequency and synaptic kinetics in the affected regions. All GIRK2 splicing isoforms examined were expressed at higher levels in the Ts65Dn in comparison to the diploid hippocampus. The pattern of GIRK2 expression in the Ts65Dn mouse brain revealed by in situ hybridization and immunohistochemistry was similar to that previously reported in the rodent brain. However, in the Ts65Dn mouse a strong immunofluorescent staining of GIRK2 was detected in the lacunosum molecular layer of the CA3 area of the hippocampus. In addition, tyrosine hydroxylase containing dopaminergic neurons that coexpress GIRK2 were more numerous in the substantia nigra compacta and ventral tegmental area in the Ts65Dn compared to diploid controls. In summary, the regional localization and the increased brain levels coupled with known function of the GIRK channel may suggest an important contribution of GIRK2 containing channels to Ts65Dn and thus to DS neurophysiological phenotypes. J. Comp. Neurol. 494:815–833, 2006. Published 2005 Wiley‐Liss, Inc.

Choline acetyltransferase and calcitonin gene-related peptide immunoreactivity in motoneurons after different types of nerve injury

SummaryThis study examined changes in choline acetyltransferase and calcitonin gene-related peptide immunoreactivity in hypoglossal motoneurons of rats at 1, 3, 7, 20 and 50 days after three types of nerve injury: crush, transection and resection. Peripheral reinnervation was assayed by retrograde labelling of the motoneurons after injections of the exogenous protein, horseradish peroxidase, into the tongue. Maximal reduction in choline acetyltransferase immunostaining occurred at seven days after nerve damage and the amount of the decrease was related to the nature of the injury. The recovery of choline acetyltransferase to normal levels was related to the timing of reinnervation after nerve crush, but not after transection or resection injuries. In contrast to these findings, a rapid increase in calcitonin gene-related peptide immunoreactivity preceded the decrease in choline acetyltransferase levels. A striking increase in calcitonin gene-related peptide immunoreactivity was observed at one day postoperative and was maximal at three days postoperatively for all injuries. Later changes in calcitonin gene-related peptide levels were dependent on the type of injury. Increased calcitonin gene-related peptide staining persisted to 20 days after nerve crush. After nerve transection or resection, calcitonin gene-related peptide immunoreactivity decreased to basal levels at seven days postoperatively. This declination was followed by a second rise in calcitonin gene-related peptide immunolabeling at 20 days for nerve transection or at 50 days after resection. Nearly complete reinnervation was established by 20 days after nerve crush. At 50 days after transection, less than half the number of normally-labelled neurons contained horseradish peroxidase. At this time only 1% of those whose axons had been resected were labelled. These observations suggest that different mechanisms regulate the responses of choline acetyltransferase and calcitonin gene-related peptide to nerve injury. The present results indicate that choline acetyltransferase levels in motoneurons can not be used to predict either the likelihood of or the timing of reinnervation after nerve transection or resection. However, our results strengthen the premise that an increase of calcitonin gene-related peptide immunoreactivity serves as a reliable index for predicting nerve regeneration/reinnervation after cranial nerve injury.

Experimental superficial siderosis of the central nervous system: Biochemical correlates

The pathogenesis of superficial siderosis of the central nervous system (CNS) may be examined by the repeated intracisternal injection of washed autologous red blood cells (RBC). In rabbits, the injections cause the accumulation of iron in the cytoplasm of microglial cells and astrocytes of cerebellar and cerebral cortices. Immunocytochemistry for ferritin reveals enhanced reaction product mainly in microglia but hemosiderin occurs only after extending the injections to 6 months. In an effort to determine the biochemical correlates of these morphological changes, iron, ferritin, ferritin subunits and the ferritin repressor protein (FRP) were quantitated. There was no increase of total iron or ferritin in the exposed cortical areas. However, the injections of RBC caused dramatic shifts of the relative contributions by heavy (H-) and light (L-) ferritin subunits. The initial response was a prompt increase of the H/L ratio to over 4.0 from the normal ratio near 1.0. Extended injections caused the ratio to drop to below unity, and the predominance of L-ferritin at 6 months coincided with the appearance of granular hemosiderin. This investigation also confirmed the presence of FRP in rabbit brain cytosols but the induction of experimental superficial siderosis did not change its levels or in vitro affinity for the iron-responsive element in ferritin messenger ribonucleic acid. It is proposed that the incrustation by hemosiderin which characterizes superficial siderosis of the CNS in humans occurs when prolonged exposure to hemoglobin produces persistent shifts of the H/L-ratios by accumulation of L-ferritin.

Intrasomatic changes in the maturing hypoglossal nucleus after axon injury

SummaryThe intrasomatic reactions to different types of peripheral nerve injury during postnatal maturation were investigated by light and electron microscopy. The hypoglossal nerve was crushed in 7 day postnatal (dpn) rats and crushed, ligated or transected in 10 and 21 day rats. Survival intervals ranged from 3 to 40 days postoperative (dpo). Normal and sham operated rats of corresponding ages served as controls. The initial intrasomatic reactions in young (7–10 dpn) rats were identical after each type of nerve injury. These reactions involved the nucleus and the perinuclear cytoplasm: severe nuclear eccentricity and elaborate infoldings of the nuclear membrane were seen. The processes of cytoplasm indenting the nuclear membrane were intensely basophilic and contained numerous polyribosomes and cisterns of rough endoplasmic reticulum (RER). The formation of organized RER was not disrupted after axonal injury. Disorganization, fragmentation and degranulation of the cisterns were not apparent until 13–20 dpo. Comparable nerve injuries to older (21 dpn) rats produced structural alterations of the same organelles. However, the initial intrasomatic response involved the organized RER and the extent of the changes was directly related to the severity of nerve injury. Nuclear changes occurred later and only after nerve ligation and transection. Therefore, two major differences characterized the intrasomatic reactions to axonal injury in young and older motoneurons. The timetable of involvement of two organelles, the nucleus and the organized RER, was reversed in the sequence of intrasomatic reactions after axonal damage during successive periods of postnatal development. The magnitude of intrasomatic reactions to different types of nerve injury was age-dependent.

Light therapy and supplementary Riboflavin in the SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis (FALS).

Familial amyotrophic lateral sclerosis (FALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and death. Mitochondrial dysfunction and oxidative stress play an important role in motor neuron loss in ALS. Light therapy (LT) has biomodulatory effects on mitochondria. Riboflavin improves energy efficiency in mitochondria and reduces oxidative injury. The purpose of this study was to examine the synergistic effect of LT and riboflavin on the survival of motor neurons in a mouse model of FALS.

Transection or electrical stimulation of the hypoglossal nerve increases glial fibrillary acidic protein immunoreactivity in the hypoglossal nucleus

Glial fibrillary acidic protein (GFAP) immunoreactivity within rat hypoglossal (XIIth) nuclei was examined 1-50 days following either unilateral nerve transection or modest electrical stimulation using indirect immunofluorescence and PAP immunohistochemistry. Both nerve transection and stimulation provoked an increase in the immunodetected GFAP within the XIIth nucleus.