Abstract Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA1 /CA3 subfields, together with reactive astrogliosis. Previous studies indicate ...that in cultured rat hippocampal neurons the Ca2+ cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca2+ chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro , in glial cells and in CA3 neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Highlights • AQP4 over-expression in perivascular astrocytes of the rat hippocampus and cortex. • IgG leakage in the hippocampal and cortical paravasal parenchyma of TMT-treated rats. • Enhanced ...neuronal VEGF/VEGFR-2 production and VEGFR-2 activation (VEGFR-2P).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Previous studies have revealed that the expression pattern of the neurokinin 1 receptor (the preferred receptor for substance P, SP) varies in different mammalian retinas. We investigated NK1 ...receptor expression in the mouse retina to provide background information for future studies in transgenic mice on SP functional roles in the retina. Mouse retinal sections were treated for single and double-label immunofluorescence. NK1 receptor immunoreactivity was in bipolar cells and in numerous amacrine cells. Double-label studies showed that NK1 receptor-expressing bipolar cells constituted a population of ON-type cone bipolar cells, since they were distinct from rod bipolar cells and contained glycine. They were nonrandomly distributed with highest density in central retina. These cells were similar and may correspond to the population of NK1 receptor-expressing bipolar cells of the rabbit retina. Different subsets of NK1 receptor-expressing amacrine cells were identified on the basis of the expression of selected neurotransmitter substances: i) about 23% of NK1 receptor-expressing amacrine cells also contained glycine; ii) the remaining 77% were likely to be GABAergic, although some inconsistency was observed in the GABA immunostaining obtained with two different GABA antibodies; iii) all dopaminergic amacrine cells also expressed NK1 receptors; iv) about one third of SP-containing amacrine cells also expressed NK1 receptors. These findings confirm and expand previous observations in rat and rabbit retinas. In particular, common to all three species is the expression of NK1 receptors in dopaminergic amacrine cells, indicating that SP neurotransmission may be a universal feature of the circuitry of the dopaminergic amacrine cell. Peculiar to the mouse retina is the presence of putative NK1 autoreceptors expressed by SP-containing amacrine cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
To complete a series of studies on the expression of substance P and neurokinin receptors in mammalian retinas, we investigated the occurrence of these molecules in developing mouse retinas and in ...retinas of mice with genetic deletion of the neurokinin 1 receptor, the preferred substance P receptor. Using semi-quantitative reverse transcription–polymerase chain reaction, we measured detectable levels of the γ isoform of preprotachykinin A (a substance P precursor) mRNA at postnatal day 4. Neurokinin 1 receptor and neurokinin 3 receptor mRNAs were also detected at postnatal day 4. While γ preprotachykinin A and neurokinin 1 receptor mRNA levels significantly increased up to eye opening (postnatal day 11), neurokinin 3 receptor mRNA levels remained constant throughout development. Substance P, neurokinin 1 receptor and neurokinin 3 receptor immunoreactivities were present at postnatal day 5. Substance P was in amacrine cells, neurokinin 1 receptor in developing amacrine and bipolar cells and neurokinin 3 receptor in OFF-type cone bipolar cells. Interestingly, a transient increase in the density of neurokinin 1 receptor immunoreactive processes was observed at eye opening in lamina 3 of the inner plexiform layer, suggesting a role of substance P and neurokinin 1 receptor in this developmental phase. However, in neurokinin 1 receptor knockout retinas, besides a significant increase of the γ preprotachykinin A mRNA levels, no major changes were detected: neurokinin 3 receptor mRNA levels as well as substance P and neurokinin 3 receptor immunostainings were similar to wild types. Together with previous studies, these observations indicate that there are major differences in neurokinin 1 receptor expression patterns among developing mammalian retinas. The observations in neurokinin 1 receptor knockout mice may not be applicable to rats or rabbits, and substance P and neurokinin 1 receptor may play different developmental roles in different species.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative ...diseases. We have previously shown that rat hippocampal neurons expressing the Ca²⁺-binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca²⁺ overload. The present study was aimed at determining whether intracellular Ca²⁺ homeostasis dysregulation is involved in the TMT-induced neurodegeneration and if intracellular Ca²⁺-buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time- and concentration-dependent Ca²⁺i increases that were primarily due to Ca²⁺ release from intracellular stores although Ca²⁺ entry through Cav1 channels also contributed to Ca²⁺i increases in the early phase of TMT action. Cell pre-treatment with the Ca²⁺ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (2 μM) significantly reduced the TMT-induced neuronal death. Moreover, CR⁺ neurons responded to TMT with smaller Ca²⁺i increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca²⁺ homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR⁺ neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca²⁺ overload.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract The specific functional and pathological alterations observed in Alzheimer's disease are less severe in the cerebellum than in other brain areas, particularly the entorhinal cortex and ...hippocampus. Since dense core amyloid-beta plaque formation has been associated with an acetylcholinesterase heterogeneous nucleator action, we examined if an acetylcholinesterase imbalance was involved in cerebellum plaque deposition. By using the canine counterpart of senile dementia of the Alzheimer's type, a promising model of human brain aging and early phases of Alzheimer's disease, we investigated how cerebellar pathology and acetylcholinesterase density could be related with cognitive dysfunction. As in Alzheimer's disease, the late affectation of the cerebellum was evidenced by its lack of amyloid-beta plaque and the presence of diffuse deposition throughout all cortical grey matter layers. The highest acetylcholinesterase optic density corresponded to cerebellar islands of the granular layer and was predominantly associated with synaptic glomeruli and the somata of Golgi cells. Its reduction correlated with aging and loss of granule cells, whereas cognitive deficit only correlated with loss of Purkinje cells. The observed Bergmann glia alterations may correspond to a reactive response to the loss and damage of the Purkinje cells, their specific neuronal partner. Regarding the role of acetylcholinesterase mediation in amyloid-beta deposition, our data argue against an interaction between these two proteins because acetylcholinesterase reduction correlates with aging but not with cognitive deficit. Finally, our data support the use of companion dogs of all breeds to study aging and early phases of Alzheimer's disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Reelin is an extracellular matrix protein which is critical for the positioning of migrating post-mitotic neurons and the laminar organization of several brain structures during development. We ...investigated the expression and localization of Reelin in the rodent peripheral nerve during postnatal development and following crush injury in the adult stage. As shown with Western blotting, immunocytochemistry and RT-PCR, Schwann cells in the developing peripheral nerve and in primary cultures from neonatal nerves produce and secrete Reelin. While Reelin levels are downregulated in adult stages, they are again induced following sciatic nerve injury. A morphometric analysis of sciatic nerve sections of reeler mice suggests that Reelin is not essential for axonal ensheathment by Schwann cells, however, it influences the caliber of myelinated axons and the absolute number of fibers per unit area. This indicates that Reelin may play a role in peripheral nervous system development and repair by regulating Schwann cell–axon interactions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In adult guinea pigs, the oculomotor nerve was sectioned proximally (at the tentorial edge) or more distally (at the orbital fissure) and immediately repaired by reapproximation. During a 24-week ...postoperative period, extrinsic eye motility was assessed by analyzing the vestibulo-ocular reflexes. The regenerated oculomotor nerve was studied morphometrically on semi-thin histological sections at 16 and 24 weeks postinjury. The selectivity of muscle reinnervation was investigated by injection of both single (horseradish peroxidase) and double (fluorescent dyes) retrograde axonal tracers into the eye muscles. Following proximal repair of the oculomotor nerve, the degree of recovery of extraocular motility varied among different animals and remained stable over long-term observations. In animals with poor recovery, aberrant eye movements were always found, and the somatotopic map of the reinnervated eye muscles was greatly altered. Distortions of the central representation were also seen in those animals in which a good level of functional recovery was seen. However, in animals with good recovery, a topographic bias was re-established by about 65% of the original neuronal population, as opposed to 26% in the animals with poor recovery. Neurons located contralateral to the axotomized nucleus sprouted intra-axially and projected their axons to denervated eye muscles. The number and diameter of the regenerated axons, the number and soma diameter of the axotomized neurons, and the ratio of distal axonal branches to proximal supporting neurons were all related to the degree of functional recovery. Following repair of the oculomotor nerve at the orbital fissure, extraocular motility had recovered in all of the animals at 16 weeks without aberrant phenomena. Functional regeneration of the distally transected oculomotor nerve is thought to be the result of selective muscle reinnervation.
Spinal cord tissue was obtained from 13- and 14-day embryonic rats and homologously grafted to the completely transected spinal cord of adult rats. Eight and 12 weeks after grafting, clinical, ...electrophysiological, histological, and neuroanatomical studies were performed. Motor performance of the hosts was assessed by the inclined-plane test. The conduction of nerve impulses across the lesion-transplantation site was evaluated by recording the spinal corticomotor and somatosensory evoked potentials. The survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin sections. The axonal interactions between the host spinal cord and the graft as well as the posttraumatic retrograde degeneration of corticospinal axons were investigated using the horseradish peroxidase (HRP) technique. Clinical and electrophysiological assessments did not demonstrate any functional activity of the graft. On histological examination, grafted neurons showed a survival rate of 55%. Such neurons exhibited a limited degree of growth and differentiation. The extent of parenchymal integration between the host spinal cord and the graft varied considerably among different specimens and in the various regions of every specimen. The HRP investigations demonstrated that some axonal interactions between the host spinal cord and the graft had occurred. Regenerated axons arising from both the spinal cord and the dorsal root ganglia of the host entered the graft and elongated in it. Also, axons from the grafted neurons were able to grow for some distance in the host spinal cord. The phenomenon of the posttraumatic retrograde degeneration of corticospinal axons was not affected by this embryonic tissue grafting.
Oculomotor nerve palsy greatly impairs the patient's daily life. After oculomotor nerve injury, when the central nerve stump is not available, neurotization of the distal nerve stump with a donor ...nerve may be performed. Here, we present an experimental anatomic study in rats related to the motor nuclear organization after facial-to-oculomotor nerve anastomosis.
In adult rats, the right oculomotor nerve was transected at the skull base. Then, the ipsilateral facial nerve was exposed at the stylomastoid foramen and connected side-to-end to one extremity of a peroneal nerve autograft. The other extremity of the nerve autograft was connected end-to-end to the distal stump of the transected oculomotor nerve. Twelve weeks later, axonal regeneration in the autograft and brainstem somatotopic representation of the reinnervated extraocular muscles were investigated by use of histological and retrograde axonal tracing techniques.
The autograft was reinnervated by a large number of small axons, 1 to 5 microm in diameter. After tracer injection into the superior rectus and medial rectus muscles, retrogradely labeled neurons were seen not only in the ipsilateral facial nucleus (16%) but also in the contralateral nucleus (8%). Labeled neurons were also seen in the ipsilateral abducens (12%), motor trigeminus (7%), trochlear (23%), and contralateral trochlear (34%) nuclei. In normal rats, the extraocular muscles are innervated by unilateral-ipsilateral brainstem motor nuclei, except for the superior rectus and superior oblique muscles, which are innervated by bilateral, primarily contralateral, nuclei.
The central rearrangement of the extraocular muscle nuclei after facial-to-oculomotor nerve anastomosis represents an original example of plasticity. Functional studies are needed to demonstrate whether this procedure might serve to restore some degree of eye motility.
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