Major histocompatibility complex class II expression by activated microglia caudal to lesions of descending tracts in the human spinal cord is not associated with a T cell response

Lesion-induced microglial/macrophage responses were investigated in post-mortem human spinal cord tissue of 20 patients who had died at a range of survival times after spinal trauma or brain infarction. Caudal to the spinal cord injury or brain infarction, a strong increase in the number of activated microglial cells was observed within the denervated intermediate grey matter and ventral horn of patients who died shortly after the insult (4–14 days). These cells were positive for the leucocyte common antigen (LCA) and for the major histocompatibility complex class II antigen (MHC II), with only a small proportion staining for the CD68 antigen. After longer survival times (1–4 months), MHC II-immunoreactivity (MHC II-IR) was clearly reduced in the grey matter but abundant in the white matter, specifically within the degenerating corticospinal tract, co-localising with CD68. In this fibre tract, elevated MHC II-IR and CD68-IR were still detectable 1 year after trauma or stroke. It is likely that the subsequent expression of CD68 on MHC II-positive microglia reflects the conversion to a macrophage phenotype, when cells are phagocytosing degenerating presynaptic terminals in grey matter target regions at early survival times and removing axonal and myelin debris in descending tracts at later survival times. No T or B cell invasion or involvement of co-stimulatory B7 molecules (CD80 and CD86) was observed. It is possible that the up-regulation of MHC II on microglia that lack the expression of B7 molecules may be responsible for the prevention of a T cell response, thus protecting the spinal cord from secondary tissue damage. Received: 12 October 1999 / Revised: 31 January 2000 / Accepted: 8 February 2000     

Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish

We analyzed pathway choices of regenerating, mostly supraspinal, descending axons in the spinal cord of adult zebrafish and the cellular changes in the spinal cord caudal to a lesion site after complete spinal transection. Anterograde tracing (by application of the tracer rostral to the spinal lesion site) showed that significantly more descending axons (74%) regenerated in the spinal gray matter of the caudal spinal cord than would be expected from random growth. Retrograde tracing (by application of the tracer caudal to the spinal lesion site) showed that, rostral to the lesion, most of these axons (80%) extended into the major white matter tracts. Thus, ventral descending tracts often were devoid of labeled axons caudal to a spinal lesion but contained many axons rostral to the lesion in the same animals, indicating a pathway switch of descending axons from the white matter to the gray matter. Ascending axons of spinal neurons were not observed regrowing to the rostral tracer application site; therefore, they most likely did not contribute to the axonal populations analyzed. A macrophage/microglia response within 2 days of spinal cord transection, along with phagocytosis of myelin, was observed caudal to the transection by immunohistochemistry and electron microscopy. Nevertheless, caudal to the lesion, descending tracts in the white matter were filled with myelin debris during the time of axonal regrowth, at least up to 6 weeks postlesion. We suggest that the spontaneous regeneration of axons of supraspinal origin after spinal cord transection in adult zebrafish may be due in part to the axons' ability to negotiate novel pathways in the spinal cord gray matter.     

Distinct types of microglial activation in white and grey matter of rat lumbosacral cord after mid-thoracic spinal transection

The inflammatory response has been characterized in the lumbosacral segments (L4-S1) of rats after spinal transection at T8. Immune cells were identified immunohistochemically using antibodies to complement type 3 receptor, CD11b (OX-42), the macrophage lysosomal antigen, CD68 (ED1), major histocompatibility complex class II (MHC II), and CD163 (ED2), a marker of perivascular cells. One week after cord transection, OX-42+ microglial density had nearly doubled. In the white matter, microglia became enlarged, often with retracted processes. In contrast, microglia in the grey matter remained ramified although nearly half of those lying medially contained clusters of ED1+ granules. After 8 weeks, ED1+ (+/-MHC II) macrophages were prominent in regions of Wallerian degeneration extending from dorsolateral to ventral funiculi. Microglial density remained raised in grey matter, particularly in the ventral horns of L4/5. Ramified microglia expressing MHC II+ (+/-ED1) extended from deep in the dorsal columns and around the central canal to the ventral columns. More ED2+ (+/-MHC II) perivascular and meningeal cells were recruited and expressed ED1. Thus, distinct from their conversion into macrophages in the white matter, the activation of ramified microglia after degeneration in the grey matter involves expression of ED1 without morphologic transformation.     

Microglia in the immune surveillance of the brain: Human microglia constitutively express HLA-DR molecules

The degree of MHC class II expression in histologically normal human brain biopsy or autopsy tissue is still controversial. According to the generally held view MHC class II expression is rather low in the normal brain with the exception of the white matter. In the present study, HLA-DR expression was examined immunocytochemically in different brain areas obtained from three autopsy cases with short post-mortem times (i.e. 6 h). Based on standard histological evaluation, the brain areas studied appeared as histologically normal tissue. In all brain areas there was a strong constitutive HLA-DR expression on ramified microglia. The number of HLA-DR-immunoreactive microglia was strongest in the white matter (the corpus callosum and the capsula interna for example). The border zone between white matter and grey matter, however, revealed a sharp contrast between a high density of HLA-DR-immunoreactive microglia in the white matter and a rather low number in the grey matter. In the grey matter, HLA-DR-immunoreactive microglia were much less frequent than in the white matter and more pronounced on perivascular cells. The staining and distribution pattern of HLA-DR-immunoreactive microglia was confirmed by immunocytochemistry with a panel of different anti-HLA-DR monoclonal antibodies as well as by quantitative analysis of the immunostaining. Unlike the HLA-DR immunoreactivity, HLA-ABC immunoreactivity (detecting MHC class I antigens) was confined to endothelia and not observed on microglia. In the choroid plexus stromal macrophages expressed both class I and II antigens (i.e. at a location which could provide the peripheral immune system access to CNS antigens). Constitutive HLA-DR expression by microglia qualifies them as the main resident antigen-presenting cell of the brain. The pronounced overall HLA-DR expression by resting microglia questions a central dogma of the brain as an immune-privileged site and further points to the key role of the microglia in brain immune surveillance.     

Sustained induction of heme oxygenase-1 in the traumatized spinal cord

Oxidative stress contributes to secondary injury after spinal cord trauma. Among the consequences of oxidative stress is the induction of heme oxygenase-1 (HO-1), an inducible isozyme that metabolizes heme to iron, biliverdin, and carbon monoxide. Here we examine the induction of HO-1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter. HO-1 was induced in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO-1 was induced in both the gray and the white matter. Thereafter, HO-1 expression was limited to degenerating fiber tracts. HSP70, a heat shock protein induced mainly by the presence of denatured proteins, was consistently colocalized with HO-1 in the microglia and macrophages. This study to demonstrates long-term induction of HO-1 and HSP70 in microglia and macrophages after traumatic injury and an association between induction of HO-1 and Wallerian degeneration. White matter degeneration is characterized by phagocytosis of cellular debris and remodeling of surviving tissue. This results in the metabolism, synthesis, and turnover of heme and heme proteins. Thus, sustained induction of HO-1 and HSP70 in microglia and macrophages suggests that tissue degeneration is an ongoing process, lasting 6 weeks and perhaps even longer.     

Non-synaptic mechanisms of Ca(2+)-mediated injury in CNS white matter.

Clinical deficits after injury to the CNS are due, in large part, to dysfunction of white matter (myelinated fiber tracts), including descending and ascending tracts in the spinal cord. A crucial set of questions, in the search for strategies that will preserve or restore function after CNS injury, centers on the pathophysiology of, and mechanisms underlying recovery of conduction in, CNS white matter. These questions are relevant both to spinal cord injury, and to brain infarction, which frequently affects white matter.     

Inhibitory proteoglycan immunoreactivity is higher at the caudal than the rostral Schwann cell graft-transected spinal cord interface

To begin to evaluate the influence that proteoglycans may have on the success of Schwann cell (SC) transplants to induce axonal regrowth across a complete transection lesion and beyond, we determined the pattern of expression of inhibitory chondroitin sulfate proteoglycans (CSPGs) 3 weeks after transplantation into completely transected adult rat thoracic spinal cord. Using immunohistochemistry, we observed that: (1) CSPGs recognized by CS-56 antibody are present on astrocytes, fibroblasts, and SCs in the distal graft, and at lesion and cystic cavity borders; (2) CS-56 immunoreactivity (IR) is greater at the caudal SC graft-host cord interface than the rostral interface; (3) phosphacan-IR, also greater at the caudal interface, is associated with astrocytes, fibroblasts, as yet unidentified cells, and extracellular matrix; (4) neurocan-IR is present on astrocytes and as yet unidentified cells in grey and white matter; and (5) NG2-IR is associated with matrix near SC grafts, unidentified cells mainly in white matter, and lesion borders and cysts. Neither oligodendrocytes nor activated macrophages/microglia were immunostained. In sum, the CSPGs studied are increased at 3 weeks, especially at the caudal SC graft-cord interface, possibly contributing to an inhibitory molecular barrier that precludes regrowing descending axons from entering the caudal host cord.     

Localization of microglia in the human fetal cervical spinal cord.

Differential morphologic subtypes of microglia have been identified in the human fetal frontal cerebrum using a lectin, Ricinus communis agglutinin 1 (RCA-1), and a monoclonal antibody, EBM-11. In this report, microglia were characterized in the human fetal cervical spinal cord. RCA-1-positive microglia were ramified in the developing gray matter while in the developing white matter they had a less differentiated (ameboid) appearance. EBM-11, a monoclonal antibody that recognizes CD68 on human macrophages, and microglia labeled only ameboid-type microglia in the developing white matter. This suggests that distinct subpopulations of microglia exist, which may represent different stages in microglial development, and that CD68 may be a differentiation marker for less mature forms. Therefore, cytologically less differentiated forms of microglia appear to be associated with myelination.     

Arrested maturation of cerebral neurons, axons and myelin: a new familial syndrome of newborns.

A new lethal familial syndrome of unknown etiology is described in two male siblings who died in the newborn period. Both had corneal edema and were hypotonic, requiring assisted ventilation at birth. Neuropathological findings included an immature appearance of neocortical neurons, with cortical architecture similar to that normally seen in an infant of 5 months gestational age. Axons and myelin were absent in the cerebral and cerebellar white matter, and also in descending white matter tracts of brainstem and spinal cord. Subacute inflammation was seen in the anterior horns of the spinal cord in both cases, although there was no evidence of inflammation elsewhere in the nervous system. Electron microscopy of endothelial cells from brain, spinal cord and a number of other tissues of the second sibling showed tubuloreticular inclusions (TRIs). There are no known previous reports of similar neuropathology. Future recognition of this condition will be important for genetic counselling.     

Differential microglial regulation in the human spinal cord under normal and pathological conditions

As the primary intrinsic immune effector cells of the central nervous system, microglia are involved in virtually all pathological processes of the brain and spinal cord including inflammatory, neurodegenerative, traumatic, neoplastic and vascular diseases. Despite this important role, there is a lack of data concerning microglial distribution and protein expression in the human spinal cord. In this study, we immunohistochemically investigated 10 normal human spinal cords to establish reference data and compared these results with 15 pathological human spinal cords deriving from distinct pathologies. Each spinal cord was evaluated at eight different levels for three white and two grey matter areas for both constitutive (MHC-II, CD68, IL-16, AIF-1, LCA, CD4) and reactive (MRP-8, MRP-14) microglial antigens. Whereas previous studies revealed significant regional differences in microglial distribution and protein expression in human brain, normal spinal cord displayed a uniform expression pattern, reaching levels of up to 17% MHC-II positive cells of the total cell population. This datum formed the basis for the further evaluation of microglia expression levels in pathological spinal cords, where levels of up to 45% positive cells were observed. Our results represent important reference values for future neuropathological diagnostic and therapeutical approaches in spinal cord pathologies.     

Magnetic Resonance Microscopy and Immunohistochemistry of the CNS of the Mutant SOD Murine Model of ALS Reveals Widespread Neural Deficits

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons and descending motor tracts of the CNS. We have evaluated the CNS of a murine model of familial ALS based on the over-expression of mutant human superoxide dismutase (mSOD; G93A) using magnetic resonance microscopy (MRM) and immunohistochemistry (IHC). Three-dimensional volumetric analysis was performed from 3D T₂*-weighted images acquired at 17.6 T at isotropic resolutions of 40 μm. Compared to controls, mSOD mice had significant reductions in the volumes of total brain, substantia nigra, striatum, hippocampus, and internal capsule, with decreased cortical thickness in primary motor and somatosensory cortices. In the spinal cord, mSOD mice had significantly decreased volume of both the total grey and white matter; in the latter case, the volume change was confined to the dorsal white matter. Increased apoptosis, GFAP positive astrocytes, and/or activated microglia were observed in all those CNS regions that showed volume loss except for the hippocampus. The MRM findings in mSOD over-expressing mice are similar to data previously obtained from a model of ALS-parkinsonism dementia complex (ALS-PDC), in which neural damage occurred following a diet of washed cycad flour containing various neurotoxins. The primary difference between the two models involves a significantly greater decrease in spinal cord white matter volume in mSOD mice, perhaps reflecting variations in degeneration of the descending motor tracts. The extent to which several CNS structures are impacted in both murine models of ALS argues for a reevaluation of the nature of the pathogenesis of ALS since CNS structures involved in Parkinson’s and Alzheimer’s diseases appear to be affected as well.     

Astrocytosis, microglia activation, oligodendrocyte degeneration, and pyknosis following acute spinal cord injury

Glial activation and degeneration are important outcomes in the pathophysiology of acute brain and spinal cord injury (SCI). Our main goal was to investigate the pattern of glial activation and degeneration during secondary degeneration in both gray matter (GM) and white matter (WM) following SCI. Adult rats were deeply anesthetized and injected with 20 nmol of N-methyl-D-aspartate (NMDA) into the ventral horn of rat spinal cord (SC) on T7. Animals were perfused after survival times of 1, 3, and 7 days. Ten-micrometer sections were submitted to immunocytochemistry for activated macrophages/microglia, astrocytes, oligodendrocytes, and myelin. Astrocyte activation was more intense in the vacuolated white matter than in gray matter and was first noticed in this former region. Microglial activation was more intense in the gray matter and was clear by 24 h following NMDA injection. Both astrocytosis and microglial activation were more intense in the later survival times. Conspicuous WM vacuolation was present mainly at the 3-day survival time and decreased by 7 days after the primary damage. Quantitative analysis revealed an increase in the number of pyknotic bodies mainly at the 7-day survival time in both ventral and lateral white matter. These pyknotic bodies were frequently found inside white matter vacuoles like for degenerating oligodendrocytes. These results suggest a differential pattern of astrocytosis and microglia activation for white and gray matter following SCI. This phenomenon can be related to the different pathological outcomes for this two SC regions following acute injury.     

Axonal loss in normal-appearing white matter in a patient with acute MS

BACKGROUND: Brain imaging studies detect abnormalities in normal-appearing white matter in patients with MS. OBJECTIVE: To investigate the histopathologic basis for these changes in autopsy tissue from a patient with MS with 9 months' disease duration and a terminal brain stem lesion. METHODS: The brain stem and spinal cord were analyzed ultrastructurally and immunocytochemically for axons, myelin, and activated microglia/macrophages. RESULTS: Pathologic findings were consistent with a terminal inflammatory demyelinated lesion at the cervicomedullary junction. The ventral spinal cord column, containing descending tracts, exhibited 22% axonal loss at segment C7, but grossly normal immunostaining for myelin. Confocal and electron microscopy revealed myelin sheaths without axonal content and initial stages of myelin degradation by activated microglia/macrophages among intact myelinated axons. Axonal number and appearance was normal in ascending sensory tracts. CONCLUSIONS: These studies confirm axonal degeneration in the absence of myelin loss as one histopathologic correlate to abnormal MR findings in patients with MS.     

Up-regulation of CD81 (target of the antiproliferative antibody; TAPA) by reactive microglia and astrocytes after spinal cord injury in the rat

We examined the expression of CD81 (also known as TAPA, or target of the antiproliferative antibody) after traumatic spinal cord injury in the rat. CD81, a member of the tetraspanin family of proteins, is thought to be involved in reactive gliosis. This is based on the antiproliferative and antiadhesive effects of antibodies against CD81 on cultured astrocytes, as well as its up-regulation after penetrating brain injury. CD81 expression following dorsal hemisection of the spinal cord was determined immunohistochemically at time points ranging from 1 day to 2 months postlesion (p.l.). In the unlesioned cord a low background level of CD81 was observed, with the exception of the ependyma of the central canal and the pia mater, which were strongly CD81-positive. One day p.l., CD81 was diffusely up-regulated in the spinal cord parenchyma surrounding the lesion site. From 3 days onward, intensely CD81-positive round cells entered the lesion site, completely filling it by 7 days p.l. Staining with the microglial markers OX-42 and Iba1 revealed that these cells were reactive microglia/macrophages. At this time, no significant CD81 expression by GFAP-positive reactive astrocytes was noted. From the second week onward, CD81 was gradually down-regulated; i.e., its spatial distribution became more restricted. The CD81-positive microglia/macrophages disappeared from the lesion site, leaving behind large cavities. After 2 months, astrocytes that formed the wall of these cavities were strongly CD81-positive. In addition, CD81 was present on reactive astrocytes in the dorsal funiculus distal from the lesion in degenerated white matter tracts. In conclusion, the spatiotemporal expression pattern of CD81 by reactive microglia and astrocytes indicates that CD81 is involved in the glial response to spinal cord injury.     

Rett syndrome: spinal cord neuropathology

The morphologic changes of the spinal cord in Rett syndrome are described in 2 young women who died at 20 and 30 years of age. Both patients had been in a severely disabled state for many years with tetraparesis and extreme muscle wasting. Degeneration and loss of spinal ganglion nerve cells, in addition to gliosis of both the white and gray matter of the spinal cord, were evident. The number of motor neurons appeared to be reduced and axonal changes suggestive of degeneration were observed in both the ascending and descending tracts.     

Subacute panencephalitis associated with chronic graft-versus-host disease

Summary A unique form of subacute panencephalitis developed in a child with aplastic anemia 8 months after an allogeneic bone marrow transplantation (BMT). It was characterized by parenchymal infiltration of CD3 lymphocytes, a marked increase in the number of microglia strongly expressing HLA-DR antigens in both the gray and white matter, and diffuse degeneration of the cerebral white matter. The onset of neurological symptoms coincided with the development of chronic systemic graft-versus-host disease (GVHD). Cellular infiltrates in the CNS lesions were exclusively CD3 lymphocytes intermingled with a small number of monocytes labeled with CD68. There was a preponderance of cells of the CD45RB phenotype. The pathological changes in visceral organs were consistent with those of chronic GVHD. In addition, scrutiny of immunohistochemistry disclosed sparse infiltration of CD3 lymphocytes and diffuse gliosis in the cerebral white matter of another child with chronic GVHD who died 9 months after allogeneic BMT. These cases are suggestive of a potential risk of CNS involvement in GVHD.Supported in part by a grant from the Ministry of Health and Welfare, Japan     

The contribution of brain stem catecholamine cell groups to the innervation of the sympathetic lateral cell column

The quantitative distributions of noradrenaline (NA), dopamine (DA) and adrenaline (A) were estimated throughout the grey and white matter at various levels of the cat spinal cord (C5, T3, T10 and L2). NA levels were high at all levels of the cord (about 1000 ng/100 mg protein), being more ventrally placed at the cervical level and tending to be more medially placed at lower thoracic and lumbar levels. DA was found at much lower concentrations than NA (< 100ng/100mg protein) and generally decreased in more caudal segments. There was a mediodorsal distribution throughout the cord. A levels were extremely low throughout the cord (< 17ng/100mg protein), but tended to be higher in the rostral-most segments.Up to 5 days after a total transection at T3, there was accumulation of NA in the white matter in regions known to contain catecholamine axon tracts. DA, however, showed a much more restricted accumulation in the white matter. The possibility of whether each catecholamine exists separately as a neurotransmitter in the cat spinal cord is discussed.     

Microglia in the human fetal spinal cord--patterns of distribution, morphology and phenotype.

Microglia, the intrinsic macrophages of the nervous system, colonise the cerebrum around the second trimester in man. In order to determine the extent of microglial influx into the nervous system, we have examined their distribution within the human fetal spinal cord in relation to astrocytic and vascular development between 9 and 16 weeks of gestation, using conventional immunohistochemistry [CD11b; CD45; CD64; CD68; ICAM-1; ICAM-2; VCAM-1; PECAM; GFAP; vimentin] and lectin histochemistry [RCA-1]. Microglia are identifiable by 9 weeks, within the ventricular/sub-ventricular zones. Human fetal microglia display heterogeneity in phenotype and are more readily identified by CD68 in the spinal cord. There is a marked influx of cells dorsal and ventral to the neural cavity, from the marginal layer [meninges/connective tissue] with advancing gestational age, with greatest cell densities towards the end of the time period in this study. This inward migration is associated with progressive vascularisation, ICAM-2 expression and co-localises with GFAP and vimentin positive radial glia. The patterns of microglial migration in human fetal cord differ from that within the cerebrum, but generally conform to a route following white to gray matter.     

Postnatal redistribution of pericruciate motor cortical projections within the kitten spinal cord

The anatomical distribution of pericruciate cortical axons in the spinal cord was examined using anterograde transport of WGA-HRP from multiple unilateral injections into defined regions of the pericruciate cortex (PC) in 20 time-mated kittens and 3 adult cats. The gray matter and descending white matter tract distributions of WGA-HRP-labelled densities were analyzed using computerized morphometry and 3-dimensional reconstruction. In kittens older than 16 days postnatal (dPN), PC axon densities were found in dorsolateral column tracts corresponding to those of the adult, indicating that the white matter projections from the PC were largely established by this age. However, in kittens less than 38-44 dPN (about 105-109 days gestation), the spinal gray matter PC axon densities were distributed widely (so as to involve dorsal, intermediate and ventral laminae, e.g., I to IX) and bilaterally at all levels of the spinal cord. This contrasted sharply with the adult spinal cord in which the majority of densities was localized to laminae IV-VII on the contralateral side. The proportion of PC densities counted in the ipsilateral grey matter of neonates was found to average 23% of the total gray matter projection, while in adults this value was 9%. In all animals, by about 44 dPN, the terminal fields became effectively restricted to the adult distribution, that is, focused predominantly in the medial portions of laminae IV-VII in the gray matter contralateral to the cortical injection site.     

Effects of selective spinal cord lesions on the spinal motor evoked potential (MEP) in the rat

The effects of selective spinal cord lesions on the motor evoked potential (MEP) in 21 rats were investigated. No significant change in peak amplitude was observed following lesions of the pyramidal tract. There was a significant decrease in peaks 1 and 2 with ventral funiculi lesions. All 4 peaks of the MEP were significantly reduced following lesions of the lateral funiculus. The most marked decrease in peak amplitude followed lateral funiculi lesions that involved the lateral grey of the spinal cord. In one animal where the lesion was confined to the grey matter in the cord there was a marked decrease in all peaks of the MEP. In 3 additional animals interruption of the descending tracts of the spinal cord via bilateral hemisections of the spinal cord failed to completely abolish the MEP. Increases in peak latency were also noted following spinal lesions. In some animals the increase in latency occurred in the absence of significant peak amplitude changes. The findings in this study refute the previously held position that the MEP in the rat arises from pyramidal tract activation. The role of the reticulospinal and propriospinal tracts in the generation and propagation of the MEP are discussed.