Alexander Werner

Neuroglial activation repertoire in the injured brain : graded response, molecular mechanisms and cues to physiological function

Damage to the central nervous system (CNS) leads to cellular changes not only in the affected neurons but also in adjacent glial cells and endothelia, and frequently, to a recruitment of cells of the immune system. These cellular changes form a graded response which is a consistent feature in almost all forms of brain pathology. It appears to reflect an evolutionarily conserved program which plays an important role in the protection against infectious pathogens and the repair of the injured nervous system. Moreover, recent work in mice that are genetically deficient for different cytokines (MCSF, IL1, IL6, TNFalpha, TGFbeta1) has begun to shed light on the molecular signals that regulate this cellular response. Here we will review this work and the insights it provides about the biological function of the neuroglial activation in the injured brain.

Integrin family of cell adhesion molecules in the injured brain: Regulation and cellular localization in the normal and regenerating mouse facial motor nucleus

Integrins are a large family of heterodimeric glycoproteins that play a crucial role in cell adhesion during development, inflammation, and tissue repair. In the current study, we investigated the localization of different integrin subunits in the mouse facial motor nucleus and their regulation after transection of the facial nerve. In the normal mouse brain, there was clear immunoreactivity for α5‐, α6‐, and β1‐integrin subunits on blood vessel endothelia and for αM‐ and β2‐subunits on resting parenchymal microglia. Facial nerve transection led to an up‐regulation of the β1‐subunit on the axotomized neurons and an increase in the α4‐, α5‐, α6‐, β1‐, αM‐, αX‐, and β2‐subunits on the adjacent, activated microglia. Quantification of the microglial integrins revealed two different expression patterns. The subunits α5 and α6 showed a monophasic increase with a maximum at day 4, the αM‐subunit a biphasic regulation, with an early peak at day 1 and an elevated plateau between day 14 and 42. At day 14, there was also an influx of lymphocytes immunoreactive for the α4β1‐ and αLβ2‐integrins, which aggregated at sites of neural debris and phagocytotic microglia. This finding was accompanied by a significant increase of the α5β1‐integrin on blood vessel endothelia. In summary, facial axotomy is followed by a strong and cell‐type–specific expression of integrins on the affected neurons and on surrounding microglia, lymphocytes, and vascular endothelia. The presence of several, strikingly different temporal patterns suggests a selective involvement of these molecules in the different adhesive events during regeneration in the central nervous system. J. Comp. Neurol. 411:162–178, 1999.

Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: Impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage‐colony stimulating factor‐deficient mice

Activation of microglia is among the first cellular changes in the injured CNS. However, little is known about their specific contribution to secondary damage or repair processes in neighboring neurons and nonneuronal cells or to the immune surveillance of the damaged tissue. Animal models with defective microglial response such as osteopetrosis provide an approach to explore these effects. Osteopetrosis (op) is an autosomal recessive mutation with a complete deficiency of the macrophage‐colony stimulating factor (MCSF; CSF‐1), an important mitogen for brain microglia. In the current study we examined the effects of this MCSF deficiency on the microglial reaction and the overall cellular response to nerve injury in the mouse axotomized facial motor nucleus. In the brain, MCSF receptor immunoreactivity was found only on microglia and was strongly up‐regulated following injury. MCSF deficiency led to a failure of microglia to show a normal increase in early activation markers (thrombospondin, MCSF receptor, αMβ2‐ and α5β1‐integrins), to spread on the surface of axotomized motoneurons, and to proliferate after injury. Early recruitment of CD3+ T‐lymphocytes to the facial nucleus 24 hours after injury was reduced by 60%. In contrast, the neuronal and astrocyte response was not affected. There was a normal increase in the neuropeptides calcitonin gene‐related peptide and galanin, neuronal c‐JUN, and NADPH‐diaphorase and a decrease in choline acetyltransferase and acetylcholinesterase. Astrocyte glial fibrillary acidic protein immunoreactivity also showed a normal increase. There was a normal influx of macrophages and granulocytes into the injured facial nerve. Synaptic stripping, neuronal survival, and speed of axonal regeneration were also not affected. The current results show a strong, selective effect of MCSF on the early activation of microglia and, indirectly, on lymphocyte recruitment. This early phase of microglial activation appears not to be involved in the process of repair following peripheral nerve injury. However, it is important in the initiation of inflammatory changes in the brain and in the interaction with the immune system. J. Comp. Neurol. 436:182–201, 2001.

Regulation of MSCF receptors on microglia in the normal and injured mouse central nervous system: A quantitative immunofluorescence study using confocal laser microscopy

The macrophage colony‐stimulating factor (MCSF) is a 40–76‐kD glycoprotein that plays an important role in the activation and proliferation of microglia both in vitro and in injured neural tissue. Here, we examined the regulation of MCSF receptor (MCSFR) and MCSF in the normal and injured mouse central nervous system (CNS) by using confocal laser microscopy, quantitative immunofluorescence, and reverse transcriptase–polymerase chain reaction (RT‐PCR) techniques.

Molecular Signals for Glial Activation: Pro- and Anti-Inflammatory Cytokines in the Injured Brain

Injury to the central nervous system leads to cellular changes not only in the affected neurons but also in adjacent glial cells. This neuroglial activation is a consistent feature in almost all forms of brain pathology and appears to reflect an evolutionarily-conserved program which plays an important role for the repair of the injured nervous system. Recent work in mice that are genetically-deficient for different cytokines (M-CSF, IL-6, TNF-alpha, TGF-beta 1) has begun to shed light on the molecular signals that regulate this cellular response. Here, the availability of cytokine-deficient animals with reduced or abolished neuroglial activation provides a direct approach to determine the function of the different components of the cellular response leading to repair and regeneration following neural trauma.

Interleukin-6 (IL6) and cellular response to facial nerve injury: effects on lymphocyte recruitment, early microglial activation and axonal outgrowth in IL6-deficient mice

Nerve injury triggers numerous changes in the injured neurons and surrounding non‐neuronal cells. Of particular interest are molecular signals that play a role in the overall orchestration of this multifaceted cellular response. Here we investigated the function of interleukin‐6 (IL6), a multifunctional neurotrophin and cytokine rapidly expressed in the injured nervous system, using the facial axotomy model in IL6‐deficient mice and wild‐type controls. Transgenic deletion of IL6 caused a massive decrease in the recruitment of CD3‐positive T‐lymphocytes and early microglial activation during the first 4 days after injury in the axotomized facial nucleus. This was accompanied by a more moderate reduction in peripheral regeneration at day 4, lymphocyte recruitment (day 14) and enhanced perikaryal sprouting (day 14). Motoneuron cell death, phagocytosis by microglial cells and recruitment of granulocytes and macrophages into injured peripheral nerve were not affected. In summary, IL6 lead to a variety of effects on the cellular response to neural trauma. However, the particularly strong actions on lymphocytes and microglia suggest that this cytokine plays a central role in the initiation of immune surveillance in the injured central nervous system.

Systemic LPS Injection Leads to Granulocyte Influx into Normal and Injured Brain: Effects of ICAM-1 Deficiency

The lipopolysaccharide (LPS) constituents of the gram-negative bacterial wall are among the most potent activators of inflammation. In the current study, we examined the effect of subcutaneous injection of Escherichia coli LPS on leukocyte influx into the normal and injured brain using endogenous peroxidase (EP). Normal brain parenchyma does not contain granulocytes and this does not change after indirect trauma, in facial axotomy. However, systemic injection of 1 mg LPS led to a gradual appearance of EP-positive parenchymal granulocytes within 12 h, with a maximum at 1-4 days after injection. Facial axotomy (day 14) led to a further 50-300% increase in granulocyte number. Of the five mouse strains tested in the current study, four--Balb/C, FVB, C57Bl/6, and C3H/N--showed vigorous granulocyte influx (60-90 cells per 20-microm section in axotomized facial nucleus, 20-40 cells per section on the contralateral side). The influx was an order of magnitude lower in the SJL mice. The peroxidase-positive cells were immunoreactive for neutrophil antigen 7/4 and alpha M beta 2 integrin, were negative for IBA1 (monocytes) and CD3 (T cells), and could be prelabeled by subcutaneous injection with rhodamine B isothiocyanate (RITC), confirming their origin as blood-borne granulocytes. All RITC-positive cells were IBA1 negative. This influx of granulocytes was accompanied by a disruption of the blood-brain barrier to albumin and induction of the cell adhesion molecule ICAM-1 on affected blood vessels. Transgenic deletion of ICAM-1 led to a more than 50% reduction in the number of infiltrating granulocytes compared to litter-matched wild-type controls, in normal brain as well as in axotomized facial motor nucleus. In summary, systemic injection of LPS leads to invasion of granulocytes into the mouse brain and a breakdown of the blood-brain barrier to blood-borne cells and to soluble molecules. Moreover, this mechanism may play a pathogenic role in the etiology of meningitis and in severe bacterial sepsis.

Regulation of the cell adhesion molecule CD44 after nerve transection and direct trauma to the mouse brain

CD44 is a cell surface glycoprotein involved in cell adhesion during neurite outgrowth, leukocyte homing, and tumor metastasis. In the current study, we examined the regulation of this molecule 4 days after neural trauma in different forms of central and peripheral injury. Transection of the hypoglossal, vagus, or sciatic nerve led to the appearance of CD44‐immunoreactivity (CD44‐IR) on the surface of the affected motoneurons, their dendrites, and their axons. Fimbria fornix transection led to CD44‐IR on a subpopulation of cholinergic neurons in the ipsi‐ and contralateral medial septum and diagonal band of Broca and colocalized with galanin‐IR. Central projections of axotomized sensory neurons to the spinal cord (substantia gelatinosa, Clarkes column) also showed an increase in CD44‐IR, which was abolished by spinal root transection. Nonneuronal CD44‐IR was mainly restricted to sites of direct injury. In the crushed sciatic nerve, CD44‐IR was found on the demyelinating Schwann cells and on infiltrating monocytes and granulocytes. Direct parasagittal transection of the cerebral cortex led to CD44‐IR on resident astrocytes and on leukocytes entering the injured forebrain tissue. CD44‐IR also increased on reactive retinal astrocytes and microglia after the optic nerve crush. Additional time points in the retina and hypoglossal nucleus (days 1, 2, and 14) and cerebral cortex (day 2) injury models also showed the same cell type pattern for the CD44‐IR. Finally, polymerase chain reaction analysis confirmed the posttraumatic expression of CD44 mRNA and detected only the standard haematopoietic CD44 splice isoform both in direct and indirect brain injury models. Overall, the current study shows the widespread, graded appearance of CD44‐IR on neurons and on nonneuronal cells, depending on the form of neural injury. Here, the ability of CD44 to bind to a variety of extracellular matrix and cell adhesion proteins and its common presence in different forms of brain pathology could suggest an important role for this cell surface glycoprotein in the neuronal, glial, and leukocyte response to trauma and in the repair of the damaged nervous system. J. Comp. Neurol. 426:468–492, 2000.

Lymphocyte infiltration in the injured brain: Role of proinflammatory cytokines

Studies using mouse axotomised facial motoneuron model show a strong and highly selective entry of CD3+ lymphocytes into the affected nucleus, with a maximum at Day 14, which coincides with the peak of neuronal cell death, microglial phagocytosis, and increased synthesis of interleukin‐1 beta (IL1β), tumour necrosis factor‐alpha (TNFα) and interferon‐gamma (IFNγ). We explored the possible involvement of these cytokines during the main phase of lymphocyte recruitment into the axotomised facial motor nucleus 7–21 days after nerve cut using mice homozygously deficient for IL1 receptor type 1 (IL1R1−/−), TNF receptor type 1 (TNFR1−/−), type 2 (TNFR2−/−) and type 1 and 2 (TNFR1&2−/−), IFNγ receptor type 1 (IFNγR1−/−), and the appropriate controls for the genetic background. Transgenic deletion of IL1R1 led to a 54% decrease and that of TNFR2 to a 44% reduction in the number of CD3+ T‐cells in the axotomised facial motor nucleus, with a similar relative decrease at Day 7, 14, and 21. Deletion of TNFR1 or IFNγR1 had no significant effect. Deletion of both TNFR1 and 2 (TNFR1&2−/−) caused a somewhat stronger, 63% decrease than did TNFR2 deletion alone, but this could be due to an almost complete inhibition of neuronal cell death. No mutations seemed to inhibit aggregation of CD3+ T‐cells around glial nodules consisting of Ca‐ion binding adaptor protein‐1 (IBA1)+ phagocytotic microglia and neuronal debris. Altogether, the current data show the importance of IL1R1 and TNFR2 as the key players during the main phase of lymphocyte recruitment to the damaged part of the central nervous system.

INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1) IN THE MOUSE FACIAL MOTOR NUCLEUS AFTER AXONAL INJURY AND DURING REGENERATION

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a widely expressed glycoprotein, which plays an important role in leukocyte extravasation and in the interaction of lymphocytes with antigen-presenting cells. In the current study we examined the regulation of ICAM-1 in the mouse facial motor nucleus after facial nerve transection, using immunohistochemistry, confocal laser microscopy and electron microscopy. In the normal facial nucleus ICAM-1 immunoreactivity was restricted to vascular endothelium. Transection of the facial nerve led to a strong and selective upregulation of ICAM-1 on activated microglia. Quantitation of microglial ICAM-1 immunoreactivity revealed a biphasic increase. The first peak 1–2 days post operation paralleling the early stage of microglial activation was followed by a decline at 4–7 days. The second induction of ICAM-1 occured at day 14 accompanying the period of neuronal cell death and microglial phagocytosis of neuronal debris. Immunoelectron microscopy showed strong ICAM-1 reactivity on the cell membrane of activated microglia at day 2. During the second peak (day 14), ICAM-1 was also observed on lymphocytes adhering to phagocytotic microglia forming aggregates around neuronal debris. No immunolabelling was observed on neurons, astrocytes or oligodendroglia. These data suggest the involvement of ICAM-1 in the adhesion of activated microglia, in their phagocytosis of neuronal debris, and also in the interaction with infiltrating lymphocytes following this injury.