Brain tumour diagnostics using a DNA methylation-based classifier as a diagnostic support tool.

AIMS: Methylation profiling (MP) is increasingly incorporated in the diagnostic process of central nervous system (CNS) tumours at our centres in The Netherlands and Scandinavia. We aimed to identify the benefits and challenges of MP as a support tool for CNS tumour diagnostics. METHODS: About 502 CNS tumour samples were analysed using (850 k) MP. Profiles were matched with the DKFZ/Heidelberg CNS Tumour Classifier. For each case, the final pathological diagnosis was compared to the diagnosis before MP. RESULTS: In 54.4% (273/502) of all analysed cases, the suggested methylation class (calibrated score ≥0.9) corresponded with the initial pathological diagnosis. The diagnosis of 24.5% of these cases (67/273) was more refined after incorporation of the MP result. In 9.8% of cases (49/502), the MP result led to a new diagnosis, resulting in an altered WHO grade in 71.4% of these cases (35/49). In 1% of cases (5/502), the suggested class based on MP was initially disregarded/interpreted as misleading, but in retrospect, the MP result predicted the right diagnosis for three of these cases. In six cases, the suggested class was interpreted as 'discrepant but noncontributory'. The remaining 33.7% of cases (169/502) had a calibrated score <0.9, including 7.8% (39/502) for which no class indication was given at all (calibrated score <0.3). CONCLUSIONS: MP is a powerful tool to confirm and fine-tune the pathological diagnosis of CNS tumours, and to avoid misdiagnoses. However, it is crucial to interpret the results in the context of clinical, radiological, histopathological and other molecular information.

Genetic variations in VEGF and VEGFR2 and glioblastoma outcome.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are central components in the development and progression of glioblastoma. To investigate if genetic variation in VEGF and VEGFR2 is associated with glioblastoma prognosis, we examined blood samples from 154 glioblastoma cases collected in Sweden and Denmark between 2000 and 2004. Seventeen tagging single nucleotide polymorphisms (SNPs) in VEGF and 27 in VEGFR2 were genotyped and analysed, covering 90% of the genetic variability within the genes. In VEGF, we found no SNPs associated with survival. In VEGFR2, we found two SNPs significantly associated to survival, namely rs2071559 and rs12502008. However, these results are likely to be false positives due to multiple testing and could not be confirmed in a separate dataset. Overall, this study provides little evidence that VEGF and VEGFR2 polymorphisms are important for glioblastoma survival.

Spontaneous regression of two putative supratentorial haemangioblastomas in one patient.

Supratentorial haemangioblastomas are exceedingly rare lesions. We report a patient with spontaneous regression of two suspected supratentorial haemangioblastomas after removal of one lesion. The patient was a 61-year-old man who had a generalised seizure. Investigation with MRI revealed three supratentorial lesions situated in the trigone, occipital and frontal locations. The lesion in the occipital area was surgically removed and the histopathology was consistent with a haemangioblastoma. MRI investigations performed 6 months and one year after the operation confirmed that the two remaining lesions had totally disappeared.

Clinicopathological phenotype of ALS with a novel G72C SOD1 gene mutation mimicking a myopathy.

A 71-year-old woman with a family history of amyotrophic lateral sclerosis (ALS) was investigated for symmetrical, proximal limb and abdominal muscle weakness. Initial examination showed mild proximal muscle weakness in the arms and legs, slightly elevated serum creatine kinase (CK) level, and normal electromyographic (EMG) findings. A myopathy was the presumed diagnosis. Over the next year, weakness became severe and tendon reflexes became unelicitable; no upper motor signs were present. EMG then showed acute and chronic denervation and a muscle biopsy showed target fibers and grouped atrophy. DNA analysis revealed a G72C CuZn-superoxide dismutase (SOD1) mutation. Fasciculations were absent throughout the disease. The patient died 53 months after symptom onset and autopsy revealed loss of lower motor neurons (LMN) and SOD1-positive inclusions. This case expands the phenotypic spectrum of ALS associated with SOD1 mutations to include presenting features that mimic a myopathy.

Heterogeneity in the expression of markers for drug resistance in brain tumors.

Brain tumors, in general, display a multidrug-resistant phenotype. This study evaluated the immunohistochemical expression and distribution of P-glycoprotein (Pgp), multidrug resistance protein (MRP1), lung resistance protein (LRP) and O6 methylguanine-DNA methyltransferase (MGMT) in low- and high-grade astrocytoma, oligodendroglioma and in different subgroups of meningioma. The results revealed a marked heterogeneity in the expression and distribution among the analyzed tumors. In astrocytoma and oligodendroglioma, Pgp and MRP1 were observed in the capillary endothelium and in scattered tumor cells, whereas LRP occurred only in tumor cells. A pronounced expression of MGMT was found independent of the histopathological grade. An enhanced expression of MRP1 and LRP in astrocytoma and oligodendroglioma were more often evident in older patients (> 50 years). Survival analysis suggested a markedly decreased overall survival for patients suffering from low-grade glioma overexpressing Pgp. In meningioma, a heterogeneous expression of Pgp, MRP1, LRP and MGMT was seen with the most prominent staining localized to the capillary endothelium. Pgp was significantly more often overexpressed (p < 0.05) in transitional compared to meningothelial meningioma. The marked heterogeneity in the expression suggests that analysis of these factors can be of importance in the selection of individualized chemotherapy, regardless of tumor type.

Neuronal loss in familial frontotemporal dementia with ubiquitin-positive, tau-negative inclusions.

The neuronal density in the frontal, temporal, and parietal lobes was determined in nine cases of familial frontotemporal dementia with ubiquitin-positive, tau-negative inclusions (FTDU). The mean age at onset was 56.9 +/- 2.2 years and the duration of disease was 6.7 +/- 0.5 years. The mean age at death was 63.6 +/- 2.2 years. There was substantial loss (34%) of brain weight (877 +/- 73 g) in the familial cases in comparison with 10 normal aged controls (1326 +/- 50 g, P < 0.001). All of the familial FTDU cases showed atrophy of the frontal, temporal, and parietal lobes; neuronal loss; vacuolation in superficial laminae; reactive astrocytosis; and ubiquitin-positive, tau-negative intracytoplasmic and intranuclear inclusions and dystrophic neurites in varying sites and numbers. Neuronal loss was estimated in nine cases of familial FTDU and in 10 aged controls using a stereological probe, the optical "disector," and a computerized stereology system (CAST-Grid, Olympus, Denmark). There was a significant reduction in neuronal density in the frontal lobe (22.3 +/- 3.8 x 10(3)/mm(3)) of familial FTDU in comparison to aged controls (33.1 +/- 1.7 x 10(3) per mm(3), P < 0.05). An estimate of the relative numbers of neurons was calculated by multiplying the numerical density by the cortical thickness, which showed a striking loss of neurons of 56% in the frontal lobe, 52% loss in the temporal lobe, and a 49% loss in the parietal lobe of familial FTDU when compared to controls. This study shows that familial FTDU has profound focal neuronal loss in multiple association areas that relate to the clinical symptoms characteristic of the disease.

Corneal endothelial integrity in mice lacking extracellular superoxide dismutase.

PURPOSE: To evaluate corneal endothelial morphology in mice without secreted extracellular superoxide dismutase (SOD) in normal ageing and in a lipopolysaccharide (LPS)-induced inflammation model and to measure the contents of SOD isoenzymes in the mouse cornea and the superoxide radical concentrations in corneas with and without extracellular SOD. METHODS: The central corneal endothelium of wild-type and extracellular SOD-null mice were studied in micrographs at eight different ages and after a unilateral intravitreal injection of LPS, with the contralateral eye serving as the control. The activities of the SOD isoenzymes in the mouse cornea were determined with a direct assay, the superoxide radical concentration was assessed by lucigenin-induced chemiluminescence, and the extracellular SOD distribution was mapped with immunohistochemistry. RESULTS: The activities of the cytosolic Cu- and Zn-containing SOD, the mitochondrial Mn-containing SOD and extracellular SOD were 4300, 15, and 340 U/g wet weight, respectively. Extracellular SOD was found in the epithelium, stroma, and endothelium. The concentration of extracellular superoxide radicals was doubled in extracellular SOD-null corneas, and the endothelial cell density decreased more with age in extracellular SOD-null than in wild-type control corneas. In the LPS-induced inflammation model, the cell density decreased more, and the cells became more irregular in extracellular SOD-null than in wild-type corneas. CONCLUSIONS: In the mouse cornea, absence of extracellular SOD leads to a higher concentration of extracellular superoxide radicals, an enhancement in the spontaneous age-related loss of endothelial cells, and an increased susceptibility to acute inflammatory endothelial damage. Extracellular SOD is likely to have a protective role in the corneal endothelium.

Vascular endothelial growth factor-A and -C protein up-regulation and early angiogenesis in a rat photothrombotic ring stroke model with spontaneous reperfusion.

This study explored the temporal expression pattern of two subtypes of vascular endothelial growth factor (VEGF) proteins and three subforms of their receptors as well as endothelial proliferation in adult rats subjected to photothrombotic ring stroke with spontaneous reperfusion in the cortical region at risk. The exposed crania of halothane-anesthetized, temperature- and blood gas-controlled male Wistar rats were irradiated with a ring laser beam started simultaneously with systemic injection of the photosensitizer erythrosin B. Rats were repeatedly injected with 5-bromodeoxyuridine (BrdU) after stroke induction. Immunohistochemistry of coronal brain sections showed that VEGF protein subtype C increased simultaneously with subtype A in the ring lesion region at 2 h after irradiation. In the cortical region at risk (i.e., the penumbra-like zone), increased VEGF-C and VEGF-A immunostaining was seen at 24 h with sustained appearance up to 72 h after ischemic onset. Correspondingly, the VEGF-C-specific receptor flt-4 and the VEGF-A receptors flt-1 and flk-1 were up-regulated in a temporal sequence similar to that of their agonist proteins in the cortical ring lesion and the region at risk. At 48 h after stroke induction, proliferating BrdU-immunopositive endothelial cells formed microvessels in the post-ischemic cortical region at risk. These vessels became more pronounced at 72 h and were still visible at 100 days after the stroke. This study suggests that VEGF-C and its receptor flt-4 may cooperate with VEGF-A and its receptors flt-1 and flk-1 to promote early angiogenesis after stroke, which may in turn contribute to spontaneous reperfusion in this focal thromboembolic stroke model.

Cortical neurogenesis in adult rats after transient middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: This study explored the possible occurrence of newly generated nerve cells in the ischemic cortex of adult rats after middle cerebral artery occlusion and reperfusion. METHODS: Nine- to 10-week-old male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion by the monofilament method. Rats received repeated intraperitoneal injections of the cell proliferation-specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Brain sections were processed for immunohistochemistry with an avidin-biotin complex-alkaline phosphatase and/or -peroxidase method. Brain sections processed with double-immunofluorescent staining were further scanned by confocal microscopy. RESULTS: Interspersed among the predominantly newly formed glial cells, some cells were double labeled by BrdU and 1 of the neuron-specific markers, Map-2, beta-tubulin III, and Neu N, at 30 and 60 days after stroke onset. These cells were randomly distributed throughout cortical layers II through VI, occurring with highest density in the ischemic boundary zone. Three-dimensional confocal analyses of BrdU and the neuron-specific marker Neu N confirmed their colocalization within the same cortical cells. CONCLUSIONS: This study suggests that new neurons can be generated in the cerebral cortex of adult rats after transient focal cerebral ischemia. Cortical neurogenesis may be a potential pathway for brain repair after stroke.

Extracellular superoxide dismutase deficiency and atherosclerosis in mice.

Lipoprotein peroxidation in the arterial wall has been implicated in atherogenesis. The superoxide radical is formed in arteries and can induce such oxidation. Extracellular superoxide dismutase (EC-SOD) occurs in high concentration in the vascular wall interstitium, and in this study, we examined the importance of the enzyme in atherogenesis. On an apolipoprotein E-null background, the limited aortic lesions induced by a 1-month atherogenic diet were larger in EC-SOD wild-type mice than in EC-SOD-null mice, whereas there were no differences between the EC-SOD genotypes in the larger lesions seen after 3 months on the diet or after 8 months on normal chow. Despite smaller or equal lesions in the EC-SOD-null mice, their cholesterol levels were somewhat higher. Also, on a wild-type background, there were no effects produced by the absence or presence of EC-SOD on atherogenic diet-induced aortic root lesions. The urinary excretion of the lipid peroxidation biomarker 8-isoprostaglandin F(2alpha) was related to the rates of atherogenesis in the mice but was not influenced by the EC-SOD genotype. Likewise, the EC-SOD status had no effect on the staining for oxidized low density lipoprotein epitopes in aortic root sections. Our findings suggest that EC-SOD has little influence on atherogenesis in mice.

Superoxide dismutase isoenzymes in the normal and diseased human cornea.

PURPOSE: The human cornea, a tissue much exposed to oxidative stress, is rich in extracellular superoxide dismutase (SOD). In this study, the contents and distributions of the SOD isoenzymes in the normal human cornea were compared with those in corneas affected by keratoconus and bullous keratopathy. METHODS: The central and peripheral parts of normal human corneas were analyzed separately. Central corneal buttons were obtained from patients with keratoconus and bullous keratopathy who were undergoing primary keratoplasty or retransplantation. SOD enzymatic activities were determined by a direct spectrophotometric method, and extracellular SOD and the cytosolic Cu- and Zn-containing SOD (CuZn-SOD) proteins were determined with ELISA and studied with immunohistochemistry. RESULTS: The total SOD content, and particularly the extracellular SOD content, was lower in the central than in the peripheral normal cornea. CuZn-SOD and extracellular SOD were demonstrated in all three corneal layers. CuZn-SOD was found in cells, whereas extracellular SOD appeared to be localized on cell surfaces, in basal membranes, and in the stroma. In keratoconus, corneal levels of extracellular SOD were half those in the control corneas, whereas CuZn-SOD and the mitochondrial Mn-containing SOD levels were normal. In bullous keratopathy, apart from edematous dilution, SOD isoenzyme levels were essentially normal. In a remarkable finding, the same pattern in SOD isoenzyme levels as in the original disease was also found at retransplantation. CONCLUSIONS: Extracellular SOD and CuZn-SOD show markedly different distribution patterns within the human cornea. Extracellular SOD activity in the central cornea is halved in keratoconus, compared with that in normal control corneas. The finding of a similar reduction at retransplantation in keratoconus suggests reduced corneal extracellular SOD synthesis in cells of the host as a cause of the low enzyme levels.

Distribution of estramustine-binding protein during postnatal development of rat brain.

Estramustine-binding protein (EMBP) is expressed in several types of brain tumors, such as astrocytoma, ependymoma, and meningioma. It binds the cytotoxic drug estramustine with high affinity and is suggested to cause accumulation of the drug in EMBP-expressing tumor cells. In this study, the spatial distribution of EMBP in normal rat brain was studied with immunohistochemistry. Brains from male and female rats of different ages were used. EMBP was found in the cytoplasm of ependymal cells, in the leptomeninges, mainly the arachnoid, and in scattered neurons. Moreover, staining was seen in nuclei of choroid plexus cells, in the granular cell layer in the cerebellum, and in a few scattered endothelial cells. The nuclear staining was more frequent in younger animals. No obvious difference in EMBP expression between male and female rats was observed. The expression of EMBP in rat brain was confirmed with nested RT-PCR. Future studies are justified to elucidate the role of EMBP-like proteins in CNS and in brain tumors.

Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats.

Clinical stroke is mostly of thromboembolic origin, in which the magnitude of brain damage resulting from arterial occlusions depends on the degree and duration of the concomitant ischemia. To facilitate more controllable and reproducible study of stroke-related pathophysiological mechanisms, a photothrombotic ring stroke model was initially developed in adult rats. The ring interior zone comprises an anatomically well confined cortical region-at-risk which is gradually encroached by progressive hypoperfusion, thus mimicking the situation (albeit in inverse fashion) of an ischemic penumbra or stroke-in-evolution. Modification of this model using a thinner ring irradiation beam resulted in late spontaneous reperfusion in the cortical region-at-risk and a remarkable morphological tissue recovery in this ostensibly critically injured region. On the other hand, doubling the thin irradiating beam intensity facilitates a complementary situation in which lack of reperfusion in the region-at-risk after stroke induction leads to tissue pannecrosis. The dual photothrombotic ring stroke model, effectuated either with or without reperfusion and thereby tissue recovery or pannecrosis, may be well suited for the study of events related to postischemic survival or cell death in the penumbra region. To popularize the photothrombotic ring stroke model, we present a detailed protocol of how this model is induced in either version as well as protocols for transcardial carbon black perfusion and laser-Doppler flowmetry experiments.

Cortical neurogenesis in adult rats after reversible photothrombotic stroke.

Neurogenesis occurs throughout life in the dentate gyrus of hippocampus and subventricular zone, but this phenomenon has rarely been observed in other brain regions of adult mammals. The aim of the current study was to investigate the cell proliferation process in the ischemically challenged region-at-risk after focal cerebral ischemia in the adult rat brain. A reversible photothrombotic ring stroke model was used, which features sustained hypoperfusion followed by late spontaneous reperfusion and a remarkable morphologic tissue recovery in the anatomically well defined somatosensory cortical region-at-risk. Twelve-week-old male Wistar rats received repeated intraperitoneal injections of the cell proliferation specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Immunocytochemistry of coronal brain sections revealed that the majority of BrdU-positive cells were of glial, macrophage, and endothelial origin, whereas 3% to 6% of the BrdU-positive cells were double-labeled by BrdU and the neuronspecific marker Map-2 at 7 and 100 days after stroke onset in the region-at-risk. They were distributed randomly in cortical layers II-VI. Three-dimensional confocal analyses of BrdU and the neuronal-specific marker Neu N by double immunofluorescence confirmed their colocalization within the same cells at 72 hours and 30 days after stroke induction. This study suggests that, as a potential pathway for brain repair, new neurons can be generated in the cerebral cortex of adult rats after sublethal focal cerebral ischemia.

Ly49A expression on T cells alters T cell selection.

Ly49 receptors are inhibitory receptors expressed on subsets of both NK cells and NK1.1(+) T cells. The function of these receptors on NK cells is believed to be important in maintaining self-tolerance, yet their role on T cells is unclear. In this report we investigated how an Ly49A transgene alters T and NK cell development in an in vivo environment, where a ligand for Ly49A is expressed. Ly49A transgenic mice that co-expressed an MHC ligand for Ly49A, H-2D(d), developed a severe inflammatory disorder that resulted in death within the first weeks of age. T cells expressing forbidden TCR V(beta) chains were found both in the thymus and periphery of transgenic mice, while non-transgenic littermates had successfully deleted these T cell subsets. These data indicate that the expression of Ly49A on T cells could alter T cell selection and allow survival of potentially self-reactive T cells.

A photothrombotic ring stroke model in rats with or without late spontaneous reperfusion in the region at risk.

This study aimed at developing a dual setup of the photothrombotic ring stroke model with or without late spontaneous reperfusion in the region at risk and to explore the morphological consequences. The exposed crania of adult male Wistar rats were subjected to a ring-shaped laser-irradiation beam (o.d. 5.0 mm, 0.35 mm thick) for 2 min simultaneously with intravenous erythrosin B (17 mg/kg) infusion. Transcardial carbon-black perfusion revealed that a laser intensity of 0.90 W/cm(2) resulted in late, that is, starting at 72 h, spontaneous reperfusion, whereas the lowest laser intensity that produced lack of reperfusion at 7 days post-irradiation was 1.84 W/cm(2). Laser-Doppler flowmetry showed prompt cortical cerebral blood flow (cCBF) reduction both in the ring lesion and region at risk (12% and 25% of control values) after high-intensity irradiation; these reduced flow values were more rapid and pronounced than in the low-intensity irradiation setup as previously shown. The high- compared with low-intensity irradiation setup produced more frequent occurrence of thrombi in the ring-lesion region and a larger ischemic cortical lesion with a more rapid pace of ischemic cellular changes in the ring-lesion region and the region at risk. The region at risk transformed into pannecrosis in the high-intensity, but recovered morphologically in the low-intensity irradiation setup. This dual photothrombotic setup with or without spontaneous reperfusion enables the study of events related to ischemic cell survival or death in an anatomically predefined region at risk.

Erythrocyte-aggregating relapsing fever spirochete Borrelia crocidurae induces formation of microemboli.

The African relapsing fever spirochete Borrelia crocidurae forms aggregates with erythrocytes, resulting in a delayed immune response. Mice were infected with B. crocidurae and monitored during 50 days after infection. Spirochetes were observed extravascularly at day 2 after infection. Two days later, inflammatory responses, cell death, and tissue damage were evident. The pathologic responses in lungs and kidneys were similar, whereas the symptoms in the brains were delayed, with a less pronounced inflammatory response. Microemboli were found in the blood vessels, possibly a result of the erythrocyte aggregation. The B. crocidurae invasion emerged more rapidly than has been described for Lyme disease-causing Borrelia species. In addition to erythrocyte rosetting, the presence of extravascular B. crocidurae indicates a novel route for these bacteria to propagate and cause damage in the mammalian host. The histopathologic findings in this study may explain the clinical manifestations of human relapsing fever.

Long-term cortical CBF recording by laser-Doppler flowmetry in awake freely moving rats subjected to reversible photothrombotic stroke.

This study aimed at developing a laser-Doppler flowmetry (LDF) device suitable for long-term cortical cerebral blood flow (cCBF) measurement in awake, freely moving rats. The device included a flow probe adapter for permanent fixation to the skull bone and a connector that held the flow probe in the adapter in exactly the same position during repeated cCBF recordings. With this LDF recording system, cCBF values were stable and unaltered in awake, freely moving rats up to 4 days after operation compared with initial recordings during anesthesia. Repeated cCBF measurements in rats after transient removal and reattachment of the flow probe revealed a coefficient of variation of 7.0-17.4%. The LDF recording system was applied to rats subjected to a photothrombotic ring stroke lesion. cCBF in the region-at-risk declined to 59-34-26-33% of baseline values (P < 0.01) at 1-2-24 48 h after irradiation with gradually restored cCBF values of 56-87% at 72-96 h post-irradiation (P < 0.01 vs. 24 h). Transcardial carbon black perfusion examination of the brains confirmed the sustained hypoperfusion in the region at risk up to 48 h post-ischemia followed by a consistently occurring late spontaneous reperfusion. In conclusion, a novel laser-Doppler cortical CBF recording system has been set up that allows stable long-term cortical CBF follow-up in awake, freely moving rats.

A photothrombotic ring stroke model in rats with remarkable morphological tissue recovery in the region at risk.

The photothrombotic ring stroke model with sustained underperfusion followed by late spontaneous reperfusion (Gu et al. 1999) was employed to study its morphological consequences. The exposed crania of adult male Wistar rats were subjected to a ring-shaped laser irradiation beam simultaneously with intravenous erythrosin B infusion. The ischemic volume was calculated from serial sections throughout the ischemic lesions at 4, 10, 24, 48, and 72 h and 7 days and 28 days after irradiation. The ischemic volume, expressed as a percentage of the ipsilateral hemispheric volume, increased steadily from 4 to 10 to 24 h to reach its maximum value at 48 h after irradiation; at 3 days, 7 days, and 28 days, the ischemic volume was reduced to 75%, 24%, and 22% of the value at 48 h. Evaluation of ischemic volumes at different anteroposterior levels revealed that the reduced ischemic volume at 72 h and later was mainly due to morphological restoration in the centrally located, nonirradiated region at risk. An initial enlargement and development of cystic coagulation necrosis was observed in the cortical areas corresponding to the ring lesion itself. In the region at risk, a gradually deteriorating neuropil and nerve cell morphology were observed over time, with maximum severity at 48 h postirradiation. At this time, most laminae II and III neurons in the region at risk exhibited eosinophilia and pyknosis but no incrustations, with small islands of less damaged neurons randomly scattered. At 72 h and up to 28 days after irradiation, these cell characteristics were no longer observed and the region at risk was well populated with neurons that had a chiefly unremarkable cytological appearance. Neuronal counts in the central part of the region at risk were performed; no significant difference in neuronal density was observed between sham-operated controls and at 28 days after irradiation. In conclusion, the consistent, late spontaneous reperfusion coincided with remarkable tissue recovery as assessed morphologically in the region at risk. The data suggest that nerve cell repair may occur even after the detection, by conventional morphological methods, of prolonged critical ischemic neuronal damage in the setting of acute ischemic stroke.

Recovery of synapses in axotomized adult cat spinal motoneurons after reinnervation into muscle.

Peripheral axotomy of adult cat spinal motoneurons induces a marked loss of synaptic boutons from the cell bodies and dendritic trees. The aim of the present study was to analyze the recovery of synaptic contacts in axotomized motoneurons following reinnervation into muscle. Adult cat spinal motoneurons were first deprived of their muscular contacts for 12 weeks and, then, allowed to reinnervate their target muscle. Two years later, regenerated motoneurons were labeled with horseradish peroxidase to allow quantitative ultrastructural analyses of the synaptic covering of the cell bodies and dendrites. Presynaptic boutons were classified according to their size and the shape of their synaptic vesicles. Results show that a recovery of synaptic covering occurs in the axotomized neurons after muscle reinnervation, but it affects various bouton types to different degrees. The number of S-type boutons synapsing with the soma was 70% higher after reinnervation than at 12 weeks after axotomy, while the number of F-type boutons had increased by only 13%. Compared with the normal situation, the number of S-type boutons synapsing with the proximal dendrites increased from 82% at 12 weeks after axotomy to 180% in the reinnervated state. In conclusion, in adult cat spinal motoneurons, the reestablishment of muscular contact is followed by a normalization of some of the synaptological changes induced by a prolonged state of axotomy. In certain respects restitution is incomplete, but in others it results in overcompensation.