Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease.

HLA-DR is a class II major histocompatibility complex antigen which in the periphery confers antigen presenting capability. We have previously shown that this marker is profusely expressed in cortex of elderly and Alzheimer's disease (AD) patients, as is the receptor for the lymphokine interleukin-2. We now report presence of additional immune-related antigens in AD, and distributional differences from normal elderly controls. In gray matter, HLA-DR immunoreactivity is normally sparse, except in AD where it co-localizes with virtually all neuritic plaques. HLA-DR positive T cells can be demonstrated in Alzheimer's disease brain tissue, as can instances of apposition between putative brain microglia and T cells. In addition, cells with the morphologic characteristics of astrocytes label for natural killer cell antigen (Leu-11), and apparent lymphocytes bearing T helper and T cytotoxic/suppressor cell antigens are observed. These and other data suggest that the glial proliferation and scavenger activity characteristic of Alzheimer's disease may occur in an immune context and may play an important role in the pathogenesis of the disorder.

Expression of fetal ALZ-50 reactive clone 1 (FAC1) in dentate gyrus following entorhinal cortex lesion.

The Fetal ALZ-50 Reactive Clone 1 (FAC1) gene is expressed at high levels during brain development and is re-expressed in some neurodegenerative diseases. It is hypothesized that FAC1 functions during neuronal differentiation and may play an active role in neuritic re-organization following brain injury. We have previously employed the entorhinal cortex lesion model to examine reactive synaptogenesis and plasticity in the hippocampal dentate molecular layer following denervating lesion. We now report re-expression of FAC1 in the molecular layer (ML) of the dentate gyrus following entorhinal cortex (ERC) lesion. Denervated hippocampi (2,6,15, and 30 days post ERC lesion) were stained with anti-FAC1 antibody and processed for both light and electron microscopy. FAC1 was rapidly re-expressed (by 2 days) following ERC lesion, paralleling our previous observations with embryonic neural cell adhesion molecule (eN-CAM). Like eN-CAM, FAC1 expression was restricted to the denervated outer ML (OML) at 2 days post lesion. Analysis of later time points revealed an elimination of FAC1 immunostaining at the inner ML (IML)/(OML) interface as IML sprouts into the denervated zone. Image analysis confirmed the diminution of FAC1 staining in the OML as the IML sprouted into the denervated zone and revealed that FAC1 expression paralleled the temporal and spatial expression of eN-CAM following ERC lesion. Ultrastructural analysis of FAC1 staining at 6 and 30 days post lesion revealed immunoreactive profiles with the morphological characteristics of dendrites and cytoplasmic staining of granule cell perikarya. Dendritic staining was localized to the denervated OML and was not associated with any other neuropil profiles within this zone; IML staining was rare and restricted to large apical dendrites proximal to granule cell perikarya. These findings suggest that re-expression of FAC1 in the denervated OML is a rapid response to brain injury and may be important in synaptic plasticity and sprouting.

Expression of differential immune factors in temporal cortex and cerebellum: the role of alpha-1-antichymotrypsin, apolipoprotein E, and reactive glia in the progression of Alzheimer's disease.

A variety of factors and processes have been implicated in the development and progression of the pathology of Alzheimer's Disease (AD), including amyloid fragment deposition, reactive gliosis, alpha-1-antichymotrypsin (ACT), and apolipoprotein E (APOE). Carriers of the APOE 4 allele have been shown to have an enhanced risk of developing AD, and the ACT signal peptide A/A genotype may modify the APOEepsilon4 risk. The protein products of these genes have been shown to enhance conversion of diffuse beta amyloid (Abeta) fibrils, which are found in diffuse plaques, to the fibrillar form found in neuritic plaques. In affected regions of AD brain, ACT and APOE colocalize with Abeta deposits and reactive microglia and astrocytes. We examined the regional distribution of ACT, APOE, and reactive glia in temporal cortex, where neuritic plaques are abundant, and cerebellum (in areas where diffuse plaques but not neuritic plaques accumulate) to examine the relationship of these markers to the deposition of Abeta. In temporal cortex, ACT and APOE staining was localized to plaque-like profiles, reactive astrocytes, and blood vessels; human leukocyte antigen-DR (HLA-DR) and glial fibrillary acidic protein (GFAP) staining revealed focal clusters of reactive microglia and astrocytes. In cerebellum, ACT and APOE immunoreactivity was never localized to plaque-like profiles but was weakly localized to unreactive astrocytes; weak HLA-DR and GFAP immunoreactivity was present on quiescent microglia throughout the cerebellum. The lack of fibrillar amyloid deposits in cerebellum, despite the presence of well-characterized markers thought to mediate the production of Abeta, suggests that this brain region may be lacking certain factors necessary for fibril formation or that the cerebellum responds differently to stimuli that successfully mediate inflammation in affected cortex.

Rapid expression and transport of embryonic N-CAM in dentate gyrus following entorhinal cortex lesion: ultrastructural analysis.

Neural cell adhesion molecules are known to be important in axon guidance and synapse formation in the developing brain. The embryonic form of neural cell adhesion molecule (eN-CAM) is reexpressed in the outer molecular layer (OML) of the dentate gyrus following entorhinal cortex (ERC) lesion. Ultrastructural analysis revealed localization of eN-CAM to the membrane of granule-cell dendritic membranes and occasionally axons within the denervated zone. Because eN-CAM is expressed rapidly (within 2 days) after ERC lesion, we were interested in the temporal sequence of expression. Denervated hippocampi (12, 15, 24, and 48 hours post-ERC lesion) were stained with anti-eN-CAM and processed for immunoelectron microscopy. At 12 hours, there was no evidence of staining for eN-CAM. By 15 hours after lesion, membranes of both dendrites and axons throughout the molecular layer exhibited moderate eN-CAM staining, and dendritic cytoplasm was heavily labeled. Twenty-four hours following lesion, plasma membrane staining of eN-CAM on both axons and dendrites had increased in intensity within the OML, whereas membrane eN-CAM staining was diminished in the inner molecular layer (IML), and the intradendritic cytoplasmic staining disappeared. By 48 hours after lesion, eN-CAM staining had disappeared from the IML but remained intense and widely distributed in the OML. These findings suggest a rapid transport of de novo synthesized protein. A generalized reaction appears to occur immediately following denervation, and eN-CAM is up-regulated in the complete expanse of the dendritic membrane, despite the fact that only the OML is denervated.(ABSTRACT TRUNCATED AT 250 WORDS)

X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.

X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-A beta antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that beta-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to lambda-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for beta-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:1223-1232, 2000)

Hypothermia attenuates the normal increase in interleukin 1 beta RNA and nerve growth factor following traumatic brain injury in the rat.

Significant morbidity and mortality associated with traumatic brain injury (TBI) are allied with secondary posttrauma inflammatory complications. Hypothermia has been suggested as a possible treatment to lessen or suppress these inflammatory reactions. We report here that interleukin 1 beta, a cytokine responsible for initiating inflammatory cascades, is elevated in rat cortex within 6 h of TBI in the rat. Nerve growth factor (NGF) RNA and protein also increased subsequently, and NGF protein remained elevated for up to 7 days. Four hours of whole body hypothermia (32 degrees C), applied immediately after the TBI, attenuated the posttrauma increase in IL-1 beta RNA and eliminated the increase in NGF RNA and protein observed in cerebral cortex following TBI. Thus, hypothermia may be an effective therapy to diminish the posttrauma inflammatory cascade in the brain (as suggested by the decrease in IL-1 beta). However, the same treatment may hinder the brain's intrinsic repair mechanisms. Optimal treatment may, therefore, require supplemental administration of neurotrophic factors or other agents along with hypothermia.

Severe controlled cortical impact in rats: assessment of cerebral edema, blood flow, and contusion volume.

Controlled cortical impact (CCI) is a contemporary model of experimental cerebral contusion. We examined the cerebrovascular and neuropathologic effects of a severe CCI in rats. The utility of magnetic resonance imaging (MRI) for the assessment of contusion volume after severe CCI was also established. Severe CCI (3.0 mm depth, 4 m/sec velocity) to the left (L) parietal cortex was produced in anesthetized (isoflurane/N2O/O2), intubated, and mechanically ventilated male Sprague-Dawley rats (n = 58). Physiologic parameters were controlled. The time course of alterations in edema [L-R% brain water (% BW) in 3-mm coronal sections through injured and contralateral hemispheres, wet-dry weight] was evaluated at 2 h, 24 h, 48 h, and 7 days posttrauma. Local cerebral blood flow (ICBF, measured in 8 structures in each hemisphere by autoradiography) was evaluated at 2 h, 24 h, and 7 days. Contusion volume (measured by histology and image analysis) was assessed at 14 days and measured in 6 rats by both MRI and histology. The survival rate after severe CCI was 96.2%. The L-R difference in % BW increased to 1.69 +/- 0.18% at 2 h, 3.00 +/- 0.08% at 24 h, 2.69 +/- 0.09% at 48 h, and 0.94 +/- 0.21% at 7 days. These values all differed from the control (p < 0.05). The % BW was greater at 24 h and 48 h than at 2 h and 7 days (p < 0.05). Marked reductions in ICBF were limited to structures in the injured hemisphere and were observed in the parietal cortex (2 and 24 h), subcortical white matter (2 and 24 h), and hippocampus (2 h), (p < 0.05) vs control rats. In the contusion core, ICBF was 19.4 +/- 8.8 mL 100 g-1 min-1 at 24 h (p = 0.011 vs normal). Necrosis was seen in large portions of the parietal cortex and subcortical white matter, and portions of the hippocampus and thalamus. Contusion volume was 47.8 +/- 9.2 mm3, which represented 14.4 +/- 2.1% of the traumatized hemisphere. Estimates of contusion volume by MRI and histology were closely correlated (r = 0.941, p < 0.017). Severe CCI in rats is accompanied by contusion, reproducible edema, and marked hypoperfusion, involving over 14% of the injured hemisphere, and can be produced with minimal mortality. T2-weighted MRI successfully and noninvasively identifies contusion volume in this model.

Epidermal growth factor receptor expression in demented elderly: localization to vascular endothelial cells of brain, pituitary and skin.

We have previously demonstrated that expression of epidermal growth factor receptor (EGFR) was upregulated on vascular endothelial cells from brains of patients with dementia but not in brains from non-demented patients. The present study used a monoclonal antibody against EGFR to examine its expression in pituitary gland, scalp, abdominal skin and brain of 29 patients with and without various dementias and neurological deficits, as well as normal aged controls. Of 20 clinically demented patients examined postmortem, 15 exhibited EGFR immunoreactivity (IR) on brain and peripheral vascular endothelial cells. Examination of nine non-demented patients revealed only 1 patient with EGFR-IR. EGFR-IR was expressed on all blood vessels, regardless of size or location within the tissue examined. Ultrastructural analysis of EGFR revealed that in pituitary, like brain, the receptor was restricted to the lumenal cell surface of vascular endothelial cells. In all cases of EGFR-IR there was an absolute correlation between central nervous system and peripheral expression; if there was EGFR-IR in brain vessels it was present in skin. If there was no staining in brain there was no peripheral skin vessel staining; and vice versa. Increased EGFR expression may indicate proliferative or regenerative changes in the vasculature of affected patients and may provide a biological marker supporting the diagnosis of dementia by analysis of skin biopsy.

Epidermal growth factor receptor expression in demented and aged human brain.

Immunoreactivity to epidermal growth factor receptor (EGFR) was present in the brain vasculature of 13/13 demented elderly patients, but absent in 9/11 non-demented elderly patients. Of the two positive non-demented patients, one had significant Alzheimer's pathology and the other spinal carcinoma. Ultrastructurally, EGFR is localized to the lumenal surface of endothelial cells.

Epidermal growth factor receptor expression in skin does not predict dementia in the elderly.

We have previously reported that epidermal growth factor receptor (EGFR) was expressed on vascular endothelial cells from postmortem specimens of demented but not cognitively normal subjects. In the present study, we examined skin biopsies from 35 living patients who were either normal or had a clinical diagnosis of probable Alzheimer's disease. In this living cohort, there was no correlation between the presence of vascular EGFR expression and clinically determined cognitive status. While a variety of factors may be responsible for these findings, endothelial EGFR expression cannot be used to confirm a premortem clinical diagnosis of dementia.

Molecular, cellular, and pathologic characterization of HLA-DR immunoreactivity in normal elderly and Alzheimer's disease brain.

Previous studies have suggested that markers of immune function may be present in brain. We have characterized one of the most important of these markers, the human major histocompatibility complex antigen HLA-DR, at molecular, cellular, and pathologic levels. The results show that an antigen with the correct molecular weight for HLA-DR and the appropriate immunoreactivity for HLA-DR monoclonal antibodies is present in nondemented elderly (ND) and Alzheimer's disease (AD) brain tissue. HLA-DR immunoreactivity is profusely expressed by brain microglia, often expressed by lymphocytes within the neuropil, rarely expressed by astrocytes, and not expressed by neurons or oligodendrocytes. Pathologically, HLA-DR-like staining in ND patients is confined primarily to white matter nonreactive or resting microglia. In AD patients, both white matter and gray matter are stained, and HLA-DR-positive reactive microglia predominate. Virtually all senile or neuritic plaques are densely HLA-DR immunoreactive: at the plaque core staining intensity is elevated as much as 50-fold, dropping to background at the plaque margin.

Structural correlates of cognition in dementia: quantification and assessment of synapse change.

Dementia results from a combination of structural and neurochemical pathologies. The most reliable index of cognition in both postmortem and biopsied AD brain is synapse loss.

Alternate strategies in lesion-induced reactive synaptogenesis: differential expression of L1 in two populations of sprouting axons.

In the CNS the cell adhesion molecule L1 plays a role in axonal growth and fasciculation. Since its roles in synapse formation and CNS regeneration are unknown, we followed the staining of L1 through the sequence of degeneration and reactive axon sprouting in the denervated outer molecular layer (ML) of the hippocampal dentate gyrus following ipsilateral entorhinal cortex (ERC) lesion. We compared immunohistological and ultrastructural localization of L1 and employed image analysis to evaluate lamina-specific changes over time. L1 staining was uniformly distributed over the ML in unlesioned animals. Following ERC lesion, L1 staining markedly declined in the outer ML; L1 staining in the inner ML remained constant. Over 30 days postlesion, commissural and associational (C/A) afferents from inner ML sprouted partway into the denervated zone, and L1 was expressed on these sprouting afferents. L1 staining exactly corresponded to fiber outgrowth as assessed by Holmes fiber stain. As the L1-bearing axons of the C/A projection expanded, staining for embryonic N-CAM (reexpressed on the dendrites of the denervated zone) appeared to recede. There was never overlap of L1 and embryonic N-CAM staining; the difference always marked the boundary between inner and outer ML. Ultrastructural analysis confirmed localization of L1 staining to axonal profiles, indicating that the new pattern of L1 staining reflected distinct types of axonal growth. These changes in cell adhesion molecule expression closely paralleled the known sequence of reactive synaptogenesis and axonal sprouting and demonstrate a link between cell adhesion molecule expression and axonal sprouting during self-repair by the CNS.

Embryonic neural cell adhesion molecule (N-CAM) is elevated in the denervated rat dentate gyrus.

We evaluated the immunohistological changes in neural cell adhesion molecule (N-CAM) expression in the adult rat dentate gyrus during the period of synaptic degeneration, axonal sprouting, and synaptogenesis following ipsilateral entorhinal cortex (ERC) lesion. This lesion denervates the outer two-thirds of the dentate granule cells dendrites and induces compensatory sprouting from the subjacent inner one-third into the denervated zone, as well as reactive synaptogenesis in the denervated outer molecular layer. In unlesioned adult hippocampus, antibodies to total N-CAM stained the inner molecular layer intensely, and the outer molecular layer (ML) more lightly. After ERC lesion the intense staining of the inner layer widened, the expansion following the known temporal sequence of commissural and associational (C/A) axon sprouting into the denervated zone. In normal unlesioned controls there was very light, uniform staining of the ML with antibodies directed against embryonic N-CAM (eN-CAM). By 2 d post-ERC lesion, the outer two-thirds of the ML stained robustly with antibody to eN-CAM. This area of intense staining receded as the C/A axon collaterals from the inner one-third entered the denervated zone, so that by 30 d the intense eN-CAM staining only occupied the outer half of the ML. The increased expression of eN-CAM remained present at 60 d post-ERC lesion, past the point that synaptic volume density has returned to normal levels in the denervated zone. Ultrastructural studies showed that the newly expressed eN-CAM was located on the surface of dendrites in the denervated zone, but was not found at the synaptic contacts.(ABSTRACT TRUNCATED AT 250 WORDS)

Up-regulation of type 2 iodothyronine deiodinase mRNA in reactive astrocytes following traumatic brain injury in the rat.

Type 2 5'-deiodinase (5'-D2), which converts thyroxine to the more active thyroid hormone 3,5,3'-triiodothyronine (T3), is believed to be an important source of intracellular T3 in the brain. The activity of this enzyme is increased in hypothyroidism and decreased in hyperthyroidism, and as such, it serves an important role to protect the brain from wide fluctuations in T3 during changes in thyroidal state. Although it has been hypothesized that T3 may facilitate neuronal regeneration after CNS injury, the 5'-D2 response to brain injury is unknown. To assess the 5'-D2 mRNA response to injury, we performed in situ hybridization following traumatic brain injury. In unlesioned animals, 5'-D2 mRNA was undetectable. At 3 days posttrauma, 5'-D2 mRNA was detected in ipsilateral cortex near the contusion. A significant further increase of 5'-D2 mRNA was noted 7 days posttrauma in both hippocampus and cortex. Similar response was also observed on the contralateral side. Colocalization of 5'-D2 mRNA with glial fibrillary acidic protein indicates that reactive astrocytes were the major cellular source for the trauma-induced 5'-D2 expression. These data demonstrate, for the first time, a trauma-induced, astrocytic up-regulation of 5'-D2 mRNA, suggesting a potential role for T3 action in adult brain's response to injury and recovery.

Traumatic brain injury-induced excitotoxicity assessed in a controlled cortical impact model.

Using a controlled cortical impact model of traumatic brain injury (TBI) coupled with tissue microdialysis, interstitial concentrations of aspartate and glutamate (together with serine and glutamine) were assessed in rat frontal cortex. Histological analysis indicated that the severity of injury following severe TBI (depth of deformation = 3.5 mm) was approximately twice that occurring following moderate TBI (depth of deformation = 1.5 mm). Both groups demonstrated significant postinjury maximal increases in excitatory amino acid (EAA) concentration, which were proportional to the severity of injury. The mean +/- SEM fold increase in dialysate concentrations of aspartate was 38 +/- 13 (n = 5) for moderate TBI and 74 +/- 12 (n = 5) for severe TBI. Fold increases in glutamate concentrations were 81 +/- 26 and 144 +/- 23 for moderate and severe TBI, respectively. Although these increases normalized within 20-30 min following moderate TBI, concentrations of aspartate and glutamate took > 60 min to normalize after severe TBI. Changes in levels of nontransmitter amino acids were much smaller. Fold increases for serine concentrations were 4.6 +/- 0.6 and 7.6 +/- 1.7 in moderate and severe TBI, respectively; glutamine concentrations had similar small fold increases (2.6 +/- 0.2 and 4.1 +/- 0.6, respectively). Calculation of interstitial concentrations following severe TBI indicated that aspartate and glutamate maximally increased to 123 +/- 20 and 414 +/- 66 microM, respectively. To determine the extent to which such tissue concentrations of EAAs could contribute to the injury seen in TBI, the EAA receptor agonists N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid were slowly injected into rat cortex. Remarkably similar histological injuries were produced by this procedure, supporting the notion that TBI is an excitotoxic injury.

Dementia associated with dorsal midbrain lesion.

Although the dorsal midbrain has been implicated in cognitive processes in animals, its role in humans is unclear. We report the neuropsychological and postmortem neuropathological findings of a 52-yr-old university professor who developed a profound dementia in association with a focal dorsal midbrain lesion. The patient's disorder appeared to result from a tuberculous granuloma based on the clinical course and autopsy results. Neuropsychologically, he exhibited a generalized impairment across most of the cognitive domains assessed. His deficits were not explained by impaired arousal, specific sensory or motor defects, depression, or hydrocephalus. Although there are inherent limitations to a single-case investigation, our observations are consistent with animal studies that have demonstrated that focal dorsal midbrain lesions may result in cognitive impairment. We propose that the dorsal midbrain is involved in cognitive processing via modulation of thalamocortical networks.

Upregulation of nerve growth factor following cortical trauma.

As part of the inflammatory response to brain injury, CSF and tissue levels of interleukin-1 beta (IL-1 beta) are elevated after trauma. This elevation in IL-1 beta initiates a cascade of events among which may be an upregulation in nerve growth factor (NGF) in brain tissue. We infused IL-1 beta into the ventricle of adult rats and found a two- to fourfold increase in NGF in the cerebral cortex, hippocampus, and cerebellum, suggesting that IL-1 beta induced in vivo may also increase NGF in the brain. To test this hypothesis we utilized two models of traumatic brain injury (TBI) in the rat and examined NGF protein and RNA in the cortex over a period of several days. Both weight drop and controlled cortical contusion models of CNS trauma demonstrated large and significant increases in NGF protein in the cortex. NGF RNA was assessed in the controlled cortical contusion model and increased approximately fivefold by 1 day post-trauma. The remarkable elevation of NGF observed following TBI suggests that its role in response to injury may be other than as a target-derived growth substance. We hypothesize that the elevation of NGF in trauma induces upregulation of enzymes which suppress free-radical formation after injury.

Astrocytes are the major source of nerve growth factor upregulation following traumatic brain injury in the rat.

Previous studies from our group have demonstrated an upregulation in nerve growth factor (NGF) RNA and protein in the cortex 24 h following traumatic brain injury (TBI) in a rat model. This increase in NGF is suppressed if rats are subjected to 4 h of whole-body hypothermia following TBI. In the present study we used in situ hybridization to extend our initial RNA gel-blot (Northern) hybridization findings by demonstrating that NGF RNA is increased in the cortex following TBI and that hypothermia diminishes this response. Further, by combining in situ hybridization with immunocytochemistry for glial fibrillary acidic protein we demonstrate that astrocytes are the major cellular source for the upregulation in NGF and that this upregulation can be observed in the hippocampus as early as 3 h posttrauma. The predominantly astrocytic origin suggests that the NGF upregulation is not related primarily to cholinotrophic activities. We hypothesize that its function is to stimulate upregulation of antioxidant enzymes, as part of an injury-induced cascade, and that supplementation of NGF or antioxidants may be warranted in hypothermic therapies for head injury.

Complement activation by beta-amyloid in Alzheimer disease.

Alzheimer disease (AD) is characterized by excessive deposition of the beta-amyloid peptide (beta-AP) in the central nervous system. Although several lines of evidence suggest that beta-AP is neurotoxic, a mechanism for beta-AP toxicity in AD brain remains unclear. In this paper we provide both direct in vitro evidence that beta-AP can bind and activate the classical complement cytolytic pathway in the absence of antibody and indirect in situ evidence that such actions occur in the AD brain in association with areas of AD pathology.