Martin Wirenfeldt

Microglia Protect Neurons against Ischemia by Synthesis of Tumor Necrosis Factor

Microglia and infiltrating leukocytes are considered major producers of tumor necrosis factor (TNF), which is a crucial player in cerebral ischemia and brain inflammation. We have identified a neuroprotective role for microglial-derived TNF in cerebral ischemia in mice. We show that cortical infarction and behavioral deficit are significantly exacerbated in TNF-knock-out (KO) mice compared with wild-type mice. By using in situ hybridization, immunohistochemistry, and green fluorescent protein bone marrow (BM)-chimeric mice, TNF was shown to be produced by microglia and infiltrating leukocytes. Additional analysis demonstrating that BM-chimeric TNF-KO mice grafted with wild-type BM cells developed larger infarcts than BM-chimeric wild-type mice grafted with TNF-KO BM cells provided evidence that the neuroprotective effect of TNF was attributable to microglial- not leukocyte-derived TNF. In addition, observation of increased infarction in TNF-p55 receptor (TNF-p55R)-KO mice compared with TNF-p75R and wild-type mice suggested that microglial-derived TNF exerts neuroprotective effects through TNF-p55R. We finally report that TNF deficiency is associated with reduced microglial population size and Toll-like receptor 2 expression in unmanipulated brain, which might also influence the neuronal response to injury. Our results identify microglia and microglial-derived TNF as playing a key role in determining the survival of endangered neurons in cerebral ischemia.

Toll-Like Receptor 2 Signaling in Response to Brain Injury: An Innate Bridge to Neuroinflammation

Reactive gliosis is a prominent feature of neurodegenerative and neuroinflammatory disease in the CNS, yet the stimuli that drive this response are not known. There is growing appreciation that signaling through Toll-like receptors (TLRs), which is key to generating innate responses to infection, may have pathogen-independent roles. We show that TLR2 was selectively upregulated by microglia in the denervated zones of the hippocampus in response to stereotactic transection of axons in the entorhinal cortex. In mice lacking TLR2, there were transient, selective reductions in lesion-induced expression of cytokines and chemokines. Recruitment of T cells, but not macrophages, was delayed in TLR2-deficient mice, as well as in mice lacking TNFR1 (tumor necrosis factor receptor 1). TLR2 deficiency also affected microglial proliferative expansion, whereas all of these events were unaffected in TLR4-mutant mice. Consistent with the fact that responses in knock-out mice had all returned to wild-type levels by 8 d, there was no evidence for effects on neuronal plasticity at 20 d. These results identify a role for TLR2 signaling in the early glial response to brain injury, acting as an innate bridge to neuroinflammation.

Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury

Reactive microgliosis is a highly characteristic response to neural injury and disease, which may influence neurodegenerative processes and neural plasticity. We have investigated the origin and characteristics of reactive microglia in the acute phase of their activation in the dentate gyrus following transection of the entorhino‐dentate perforant path projection. To investigate the possible link between microglia and hematopoietic precursors, we analyzed the expression of the stem cell marker CD34 by lesion‐reactive microglia in conjunction with the proliferation marker bromodeoxyuridine (BrdU) and the use of radiation bone marrow (BM) chimeric mice. We found that CD34 is upregulated on early‐activated resident microglia, rather than by infiltrating bone marrow‐derived cells. The number of CD34+ microglia peaked at day 3 when 67% of the resident CD11b/Mac‐1+ microglia co‐expressed CD34, and all CD34+ cells co‐expressed Mac‐1, and decreased sharply toward day 5, unlike Mac‐1, which was maximally expressed at day 5. Approximately 80% of the CD34+ cells in the denervated dentate gyrus had incorporated BrdU into their nuclei at day 3. We also showed that CD34 is upregulated on early‐activated microglia in the facial motor nucleus following peripheral axotomy. The results suggest lesion‐reactive microglia to consist of functionally distinct subpopulations of cells; a major population of activated resident CD34+Mac‐1+ microglia with a high capacity for self‐renewal, and a subpopulation of CD34−Mac‐1+ microglia which has a mixed extrinsic and intrinsic origin and whose proliferative capacity is unknown.

Estimation of absolute microglial cell numbers in mouse fascia dentata using unbiased and efficient stereological cell counting principles

Stereology offers a set of unbiased principles to obtain precise estimates of total cell numbers in a defined region. In terms of microglia, which in the traumatized and diseased CNS is an extremely dynamic cell population, the strength of stereology is that the resultant estimate is unaffected by shrinkage or expansion of the tissue. The optical fractionator technique is very efficient but requires relatively thick sections (e.g., ≥20 μm after coverslipping) and the unequivocal identification of labeled cells throughout the section thickness. We have adapted our protocol for Mac‐1 immunohistochemical visualization of microglial cells in thick (70 μm) vibratome sections for stereological counting within the murine hippocampus, and we have compared the staining results with other selective microglial markers: the histochemical demonstration of nucleotide diphosphatase (NDPase) activity and the tomato lectin histochemistry. The protocol gives sections of high quality with a final mean section thickness of >20 μm (h = 22.3 μm ± 0.64 μm), and with excellent rendition of Mac‐1+ microglia through the entire height of the section. The NDPase staining gives an excellent visualization of microglia, although with this thickness, the intensity of the staining is too high to distinguish single cells. Lectin histochemistry does not visualize microglia throughout the section and, accordingly, is not suited for the optical fractionator. The mean total number of Mac‐1+ microglial cells in the unilateral dentate gyrus of the normal young adult male C57BL/6 mouse was estimated to be 12,300 (coefficient of variation (CV) = 0.13) with a mean coefficient of error (CE) of 0.06. The perspective of estimating microglial cell numbers using stereology is to establish a solid basis for studying the dynamics of the microglial cell population in the developing and in the injured, diseased and normal adult CNS.

Differences in Origin of Reactive Microglia in Bone Marrow Chimeric Mouse and Rat After Transient Global Ischemia.

Current understanding of microglial involvement in disease is influenced by the observation that recruited bone marrow (BM)-derived cells contribute to reactive microgliosis in BM-chimeric mice. In contrast, a similar phenomenon has not been reported for BM-chimeric rats. We investigated the recruitment and microglial transformation of BM-derived cells in radiation BM-chimeric mice and rats after transientglobal cerebral ischemia, which elicits a characteristic microglialreaction. Both species displayed microglial hyperplasia and rod cell transformation in the hippocampal CA1 region. In mice, a subpopulation of lesion-reactive microglia originated from transformed BM-derived cells. By contrast, no recruitment or microglial transformation of BM-derived cells was observed in BM-chimeric rats. These results suggest that reactive microglia in rats originate from resident microglia, whereas they have a mixed BM-derived and resident origin in mice, depending on the severity of ischemic tissue damage.

Up-regulation of PK11195 binding in areas of axonal degeneration coincides with early microglial activation in mouse brain

Increased binding of the peripheral benzodiazepine binding site (PBBS) ligand [3H]PK11195 in the central nervous system of patients suffering from acute and chronic neuropathology has been associated with reactive microgliosis. However, it remains uncertain which stages of microglial activation occur in conjunction with the increased [3H]PK11195 binding. We used quantitative autoradiography for [3H]PK11195 and quantitative polymerase chain reaction for PBBS mRNA and markers of early and late microglial activation to investigate the time‐course of cellular responses in the hippocampus of mice with degeneration of the entorhinal‐hippocampal perforant path. The axonal lesion evoked an increase in the Bmax for [3H]PK11195 in hippocampus which peaked at 2 days post‐lesion, remained elevated at day 5 and began to decline at 10 days post‐lesion. These changes occurred in the absence of significant changes in affinity in vitro. Quantitative polymerase chain reaction analysis of isolated hippocampi using exon‐specific primers indicated the presence of several splice variants of PBBS mRNA, which appeared to be affected differentially by the lesion. The changes in PBBS mRNA and CD11b mRNA levels correlated with the Bmax for [3H]PK11195 during 10 days post‐lesion, suggesting that microglial activation couples with increases in mRNA levels for these markers. In addition, the onset of changes in PBBS mRNA levels coincided with the significantly elevated tumor necrosis factor mRNA levels present during early microglial activation at 2 days post‐lesion. We conclude that up‐regulation of [3H]PK11195 binding and PBBS mRNA levels coincided with early microglial activation, characterized by concomitantly increased microglial tumor necrosis factor mRNA levels, and persisted throughout the period with reactive microgliosis.

Tumor necrosis factor and its p55 and p75 receptors are not required for axonal lesion-induced microgliosis in mouse fascia dentata

Tumor necrosis factor (TNF) is a potent pro‐inflammatory and neuromodulatory cytokine. In the CNS it is produced primarily by microglia and considered to regulate microglial activation. On the basis of previous observations of increased microglial TNF mRNA synthesis in areas of anterograde axonal and terminal degeneration in mice, we studied the effect of TNF and its p55 and p75 receptors on axonal lesion‐induced microglial activation in fascia dentata following transection of the perforant path (PP) projection. Unexpectedly, cell counting showed that the axonal lesion‐induced microglial response in TNF and TNF‐p55p75 receptor knock out mice and C57BL/6 mice was similar 5 days after the lesion. In addition, the microglial expression of the lysosomal‐associated antigen CD68, and the clearance of MBP+ myelin debris appeared similar in TNF and TNF‐p55p75 receptor knock out mice compared to C57BL/6 mice. Quantitative PCR and in situ hybridization showed the expression of TNF mRNA to be maximally upregulated 6 h after the lesion, and confirmed that TNF mRNA was still upregulated 5 days after lesion when microglial numbers, CD11b mRNA level, and cellular TNF‐p55 and ‐p75 receptor mRNA level reached maximum. However, in spite of the induction of TNF mRNA, TNF protein level remained at base‐line in fascia dentata using immunohistochemistry and ELISA. In conclusion, the results showed a lower than expected lesion‐induced increase in TNF protein, and that neither TNF nor its receptors were required for the axonal lesion‐induced microglial morphological transformation and proliferation or for the initial clearance of degenerated myelin in the PP‐deafferented fascia dentata.

Quantification of Microglial Proliferation and Apoptosis by Flow Cytometry

Microglia are innate immune cells that survey the central nervous system (CNS) and respond almost immediately to any disturbance in CNS homeostasis. They are derived from primitive yolk sac myeloid progenitors and in the mouse colonize the CNS during fetal development. As a population, microglia have the potential to expand rapidly in response to inflammatory stimuli, injury, or any other pathological changes, due to a high capacity for proliferation. In addition, apoptotic mechanisms can be evoked to retract the microglial population, as reactivity declines. In the normal CNS, a low rate of proliferation and apoptosis maintain a low rate of microglial turnover. Here, we describe quantitative analysis of proliferation and apoptosis of microglial cells isolated from individual adult mice by flow cytometry, which allows distinction from perivascular or infiltrating macrophages, based on differential expression of CD45. These methods can be applied to analyze microglial turnover in various models of neuroinflammation.

Asymptomatic loss of intraepidermal nerve fibers with preserved thermal detection thresholds after repeated exposure to severe cold

Cold‐induced peripheral neuropathy has been described in individuals exposed to severe cold resulting in pain, hypersensitivity to cold, hyperhidrosis, numbness, and skin changes. Nerve conduction studies and thermal detection thresholds are abnormal in symptomatic patients, and intraepidermal nerve fiber density (IENFD) in skin biopsies is reduced.

Hereditary spastic paraplegia type 8: Neuropathological findings

An interesting report is presented by de Bot et al (2012) (4) on a rare type 8 autosomal-dominant form of hereditary spastic paraplegia (HSP) caused by a mutation in the VCP gene. Here, we present, for the first time we believe, the neuropathological findings in an HSP type 8 (SPG8) case with a confirmed KIAA0196 mutation. HSP is clinically and genetically a heterogeneous group of disorders with more than 72 loci and 55 genes known to be involved so far (12). SPG8 has been described in 13 families (2, 5, 8, 10) and presents as a pure spastic paraplegia without associated neurological abnormalities in all families but one (2). SPG8 is caused by mutations in the strumpellin gene KIAA0196. Eleven different missense mutations (2, 5, 10) and one large deletion in intron 10— exon 15 of KIAA0196 (10) have been identified so far in patients with SPG8 (www.hgmd.cf.ac.uk). Strumpellin knockdown experiments in human neuroblastoma cells demonstrated reduced axonal outgrowth, and knockdown studies in zebrafish revealed severe contractile cardiac dysfunction, tail curvature, and impaired motility, implying a strumpellin loss-of-function pathogenesis of SPG8 (3). The major neuropathological finding in pure HSP is axonal degeneration, maximal in the terminal portions of the longest descending and ascending tracts (1). A case-control study quantifying corticospinal tract axon number showed reduced cross-sectional area and axonal density in the corticospinal tracts with a more pronounced axonal loss in the distal neuraxis in HSP patients. The cross-sectional area and axonal density of the sensory tracts were reduced only in the upper regions of the spinal cord. This is consistent with a “dying back” axonal degeneration (6). The proband presented here is a 46-year-old male with a slowly progressing spastic paraplegia. He was healthy until the age of 37, where he had onset of pain in the lower limbs, intermittent dropfoot, foot clonus, and slight ataxia. At age 45, he had an unaided walking distance of 200 m, spasticity and exaggerated deep tendon reflexes in the lower limbs, foot clonus and slight paresis of the hip corresponding to an estimated Spastic Paraplegia Rating Scale (SPRS) score of 19. He had no symptoms from the bladder, and no cognitive or psychiatric symptoms. MRI of the brain and spinal cord was normal. The proband’s mother was healthy until her first pregnancy at 25 years of age, where she exhibited slight paresis and spasticity of the legs. A neurological examination at age 34 revealed slight eye movement ataxia, ataxia of the legs, and exaggerated patellar and heel tendon reflexes corresponding to an estimated SPRS score of 15. She was suspected of having multiple sclerosis, but the diagnosis was never confirmed. She was re-evaluated at age 42 because of urine incontinence and progression of the spastic paraparesis— signs and symptoms corresponding to an estimated SPRS score of 20. At the age of 61, she suffered from severe depression and complained of cognitive deficits. Her unaided walking distance was 25 m (estimated SPRS score 25). She died 70-year old following a hemicolectomy caused by stage T3 colon cancer. Her medical history also included surgical treatment and radiotherapy for advanced breast cancer, hyperthyroidism, and atrial fibrillation. An autopsy was performed to determine the cause of the patient’s symptoms. Informed consent for performing the autopsy and related analyses as well to publish this article was obtained from the son of the deceased woman. There are no other known affected family members. A 43-yearold brother of the proband has no symptoms of spastic paraplegia. The maternal grandfather died suddenly and unexpectedly 65-year old and the maternal grandmother had unconfirmed Parkinson’s disease and died 89-year old. Mutational analysis of genomic DNA from a peripheral blood sample from the proband revealed a missense mutation in the gene for strumpellin (KIAA0196) in exon 16: c.1876G>T; p.(Val626Phe) previously found to be heterozygously present in a British family and three large North American families with European ancestry thus confirming the clinical diagnosis of SPG8. The presence of the KIAA0196 mutation in the proband’s deceased mother was confirmed on DNA extracted from frozen normal colon tissue. The mutation was absent in DNA extracted from peripheral blood from the proband’s younger brother. The weight of the fixated brain was 1002 grams. The gross external appearance of the brain was normal and there were no signs of atrophy. Sections from the cerebrum, cerebellum, pons, mesencephalon and medulla oblongata appeared normal, without signs of atrophy, which was confirmed by histological examination. The spinal cord was 39-cm long. There was severe atrophy of the spinal cord between the cervical and lumbar enlargements, where the spinal cord consisted mainly of vessels and meninges (Figure 1). Humped surfaces were found corresponding to the cervical enlargement and more widespread above, at, and below the lumbar enlargement. Grey matter was not readily distinguished on gross