Reactions of the rat musculoskeletal system to compressive spinal cord injury (SCI) and whole body vibration (WBV) therapy.

Traumatic spinal cord injury (SCI) causes a loss of locomotor function with associated compromise of the musculo-skeletal system. Whole body vibration (WBV) is a potential therapy following SCI, but little is known about its effects on the musculo-skeletal system. Here, we examined locomotor recovery and the musculo-skeletal system after thoracic (T7-9) compression SCI in adult rats. Daily WBV was started at 1, 7, 14 and 28 days after injury (WBV1-WBV28 respectively) and continued over a 12-week post-injury period. Intact rats, rats with SCI but no WBV (sham-treated) and a group that received passive flexion and extension (PFE) of their hind limbs served as controls. Compared to sham-treated rats, neither WBV nor PFE improved motor function. Only WBV14 and PFE improved body support. In line with earlier studies we failed to detect signs of soleus muscle atrophy (weight, cross sectional diameter, total amount of fibers, mean fiber diameter) or bone loss in the femur (length, weight, bone mineral density). One possible explanation is that, despite of injury extent, the preservation of some axons in the white matter, in combination with quadripedal locomotion, may provide sufficient trophic and neuronal support for the musculoskeletal system.

[Regeneration of the facial nerve in comparison to other peripheral nerves : from bench to bedside]. / Regeneration des N. facialis im Vergleich zu anderen peripheren Nerven : Aktuelles aus der Forschung für die Klinik.

Despite increasing knowledge of cellular and molecular mechanisms determining the success or failure of peripheral nerve regeneration, no effective treatments for peripheral nerve injury exist. Newly developed and validated approaches for precise numerical assessment of motor deficits have recently allowed testing of novel strategies in experimental animals. One of these approaches is the daily manual stimulation of the denervated musculature. This treatment is effective in cases of cranial nerve lesions with preservation of the sensory input (facial or hypoglossal nerve) and has the potential of direct translation in clinical settings. However, manual stimulation appears to be ineffective for the treatment of mixed peripheral nerve injuries. Generally, no long-term improvement of functional recovery is achieved by electrical stimulation in rodents. While short-term post-traumatic stimulation of the proximal nerve stump has no negative effects, direct electrical stimulation of the muscle during the period of de- and reinnervation appears to hinder muscle fibre reinnervation. Finally, experimental evidence suggests that application of peptides known as glycomimetics, which mimic functional properties of carbohydrate molecules, may provide significant benefits after injuries of mixed peripheral nerves.

Experimental and theoretical studies of sequence effects on the fluctuation and melting of short DNA molecules.

Understanding the melting of short DNA sequences probes DNA at the scale of the genetic code and raises questions which are very different from those posed by very long sequences, which have been extensively studied. We investigate this problem by combining experiments and theory. A new experimental method allows us to make a mapping of the opening of the guanines along the sequence as a function of temperature. The results indicate that non-local effects may be important in DNA because an AT-rich region is able to influence the opening of a base pair which is about 10 base pairs away. An earlier mesoscopic model of DNA is modified to correctly describe the timescales associated with the opening of individual base pairs well below melting, and to properly take into account the sequence. Using this model to analyze some characteristic sequences for which detailed experimental data on the melting is available (Montrichok et al 2003 Europhys. Lett. 62 452), we show that we have to introduce non-local effects of AT-rich regions to get acceptable results. This brings a second indication that the influence of these highly fluctuating regions of DNA on their neighborhood can extend to some distance.

Crosslinking proteins to nucleic acids by ultraviolet laser irradiation.

Ultraviolet (UV) irradiation can initiate complex formation between proteins and DNA or RNA and so can be used to study such interactions. However, crosslink formation by standard UV light sources can take up to several hours. More recently, a beam of monochromatic UV light from a laser has been used to initiate crosslinking in nano- and picosecond time intervals. As noted in an earlier TIBS article 'the advantages of short pulse times and high-energy fluxes should make this a valuable technique in the future'. In this review we characterize laser-induced crosslinking and explore the applications of this method.

Manual stimulation of forearm muscles does not improve recovery of motor function after injury to a mixed peripheral nerve.

Transection and re-anastomosis of the purely motor facial nerve leads to poor functional recovery. However, we have recently shown in rat that manual stimulation (MS) of denervated vibrissal muscles reduces the number of polyinnervated motor endplates and promotes full recovery of whisking. Here, we examined whether MS of denervated rat forearm muscles would also improve recovery following transection and suture of the mixed (sensory and motor) median nerve (median-median anastomosis, MMA). Following MMA of the right median nerve, animals received no postoperative treatment, daily MS of the forearm muscles or handling only. An almost identical level of functional recovery, measured by the force of grip in grams, was reached in all animals by the sixth postoperative week and maintained till 3 months following surgery regardless of the postoperative treatment. Also, we found no differences among the groups in the degree of axonal sprouting, the extent of motor endplate polyinnervation and in the soma size of regenerated motoneurons. Taken together, we show that while MS is beneficial following motor nerve injury, combined strategies will be required for functional recovery following mixed nerve injury.

[Experimental studies for the improvement of facial nerve regeneration]. / Experimentelle Untersuchungen zur Verbesserung der Fazialisregeneration.

Using a combination of the following, it is possible to investigate procedures to improve the morphological and functional regeneration of the facial nerve in animal models: 1) retrograde fluorescence tracing to analyse collateral axonal sprouting and the selectivity of reinnervation of the mimic musculature, 2) immunohistochemistry to analyse both the terminal axonal sprouting in the muscles and the axon reaction within the nucleus of the facial nerve, the peripheral nerve, and its environment, and 3) digital motion analysis of the muscles. To obtain good functional facial nerve regeneration, a reduction of terminal sprouting in the mimic musculature seems to be more important than a reduction of collateral sprouting at the lesion site. Promising strategies include acceleration of nerve regeneration, forced induced use of the paralysed face, mechanical stimulation of the face, and transplantation of nerve-growth-promoting olfactory epithelium at the lesion site.

Persistent interactions of core histone tails with nucleosomal DNA following acetylation and transcription factor binding.

In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

Involvement of retinoblastoma protein and HBP1 in histone H1(0) gene expression.

The histone H1(0)-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H1(0) promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H1(0) gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H1(0) H4 box were therefore expected to link differentiation-dependent expression of H1(0) to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H1(0) H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H1(0) gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H1(0), HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells.

Early rehabilitation model shows positive effects on neural degeneration and recovery from neuromotor deficits following traumatic brain injury.

This study used an experimental early rehabilitation model combining an enriched environment, multisensory (visual, acoustic and olfactory) stimulation and motor training after traumatic brain injury (via fluid-percussion model) to simulate early multisensory rehabilitation. This therapy will be used by brain injured patients to improve neural plasticity and to restore brain integration functions. Motor dysfunction was evaluated using a composite neuroscore test. Direct structural effects of traumatic brain injury were examined using Fluoro-Jade staining, which allows identification of degenerating neural cell bodies and processes. Animals in the rehabilitation model group performed significantly better when tested for neuromotor function than the animals in standard housing in the 7-day and 15-day interval after injury (7d: p=0.005; 15d: p<0.05). Statistical analysis revealed significantly lower numbers of Fluoro-Jade positive cells (degenerating neurons) in the rehabilitation model group (n=5: mean 13.4) compared to the standard housing group (n=6: mean 123.8) (p<0.005). It appears that the housing of animals in the rehabilitation model led to a clear functional increase in neuromotor functions and to reduced neural loss compared with the animal group in standard housing.

Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex binding.

Here we have examined HIV-1 nucleosome remodeling upon the binding of transcription factors and the SWI/SNF complex using a novel approach. The approach combines UV laser protein-DNA crosslinking, electrophoretic mobility-shift analysis and DNase I protection analysis with immunochemical techniques. It was found that single activator-bound HIV-1 nucleosomes exhibit very weak perturbation in histone NH(2) tail-DNA interactions. However, the simultaneous binding of the transcription activators Sp1, NF-kB1, LEF-1 and USF synergistically increased the release of histone NH(2) tails from nucleosomal DNA. In contrast, the binding of SWI/SNF complex to HIV-1 nucleosome disrupted structured histone domain-DNA contacts, but not histone NH(2) tail-DNA interactions. Stable remodeled nucleosomes, (obtained after detachment of SWI/SNF), displayed identical structural alterations with those bound to SWI/SNF. These results demonstrate a different in vitro remodeling of the HIV-1 nucleosome upon the binding of multiple transcription activators and of SWI/SNF complex.

Axonal branching and recovery of coordinated muscle activity after transection of the facial nerve in adult rats.

Facial nerve surgery inevitablyleads to pareses, abnormally associated movements, and pathologically altered reflexes. The reason for this "post-paralytic syndrome" is the misdirected reinnervation of targets, which consists of two major components. First, due to malfunctioning axonal guidance, a muscle gets reinnervated by a "foreign" axon, that has been misrouted along a "wrong" fascicle. Second, the supernumerary collateral branches emerging from all transected axons simultaneously innervate antagonistic muscles and cause severe impairment of coordinated activity. Since it is hardly possible to influence the first major component and improve the guidance of several thousands of axons, we concentrated on the second major component and tried to reduce the collateral axonal branching. The efficiency of various treatments was evaluated in rats by determining: (1) the degree of post-operative axonal branching as estimated by the number of double-or triple-labeled perikarya after application of crystalline DiI, Fluoro-Gold (FG), and Fast Blue (FB) to the zygomatic, buccal, and marginal mandibular branch of the facial nerve respectively; (2) the accuracy of reinnervation as estimated by the number of double-labeled perikarya innervating the whisker pad muscles before and after surgery as shown by intramuscular injections of FG and FB respectively; (3) the recovery of vibrissal motor performance, estimated by a video based motion analysis. So far, we have tried to reduce branching by alteration of the afferent trigeminal input to the axotomized facial motoneurons and by focal application of: (1) neurite outgrowth fostering ECM proteins; (2) neutralizing antibodies to NGF, BDNF, CNTF, GDNF, IGF-I, and FGF-II; (3) suspensions of olfactory ensheathing cells, Schwann cells, and bone marrow stroma cells; and (4) pieces of autologous olfactory mucosa to the transection site. Although most of these manipulations do influence peripheral nerve regeneration to some extent, only the application of autologous olfactory mucosa yielded a major improvement, i.e., better function.

Long-term effects of enriched environment on neurofunctional outcome and CNS lesion volume after traumatic brain injury in rats.

To determine whether the exposure to long term enriched environment (EE) would result in a continuous improvement of neurological recovery and ameliorate the loss of brain tissue after traumatic brain injury (TBI) vs. standard housing (SH). Male Sprague-Dawley rats (300-350 g, n=28) underwent lateral fluid percussion brain injury or SHAM operation. One TBI group was held under complex EE for 90 days, the other under SH. Neuromotor and sensorimotor dysfunction and recovery were assessed after injury and at days 7, 15, and 90 via Composite Neuroscore (NS), RotaRod test, and Barnes Circular Maze (BCM). Cortical tissue loss was assessed using serial brain sections. After day 7 EE animals showed similar latencies and errors as SHAM in the BCM. SH animals performed notably worse with differences still significant on day 90 (p<0.001). RotaRod test and NS revealed superior results for EE animals after day 7. The mean cortical volume was significantly higher in EE vs. SH animals (p=0.003). In summary, EE animals after lateral fluid percussion (LFP) brain injury performed significantly better than SH animals after 90 days of recovery. The window of opportunity may be wide and also lends further credibility to the importance of long term interventions in patients suffering from TBI.

Functional recovery after experimental spinal cord compression and whole body vibration therapy requires a balanced revascularization of the injured site.

PURPOSE: Based on several positive effects of whole-body-vibration (WBV) therapy on recovery after SCI, we looked for correlations between functional (analysis of locomotion), electrophysiological (H-reflex) and morphological (density of functioning capillaries) measurements after SCI and WBV-treatment. METHODS: Severe compression SCI at low-thoracic level (T8) in adult female Wistar rats was followed by WBV twice a day (2 × WBV) over a 12-week post-injury period. Intact rats and rats with SCI but no WBV-therapy ("No-WBV") served as controls. Recovery of locomotion was determined by BBB-locomotor rating, foot stepping angle (FSA), rump-height index (RHI), correct ladder steps (CLS) and H-reflex at 1, 3, 6, 9, and 12 weeks after SCI. Animals were sacrificed by an overdose of Isoflurane (Abbott). One hour later their spinal cords were fixed in 4% PFA for 24 h. Samples from the thoracic cord containing the lesion site and from the lumbar intumescence were cut into 10 µm thick longitudinal frozen sections. RESULTS: All functioning capillaries were unequivocally identified because the endogenous peroxidase of the erythrocytes was clearly visualized with 0.05% diaminobenzidine (DAB). A determination of their absolute (in µm2) and proportional areas (percent of photographed tissue) revealed a significantly denser capillary network in the WBV-treated rats: 1,66 ± 0,41% in the "vibrated" rats versus 0,79 ± 0,19% in the "No-WBV" animals. The portion of the capillary network in intact rats was 1,51 ± 0,69%. Surprisingly, even though the vascularization in the treated animals was significantly increased, this had no beneficial influence on the recovery of functions after SCI. CONCLUSION: The results of this study provide for the first time evidence that intensive WBV-therapy leads to a significantly denser capillary network in the lesioned spinal cord. However, since this higher capillary density is not associated with improved functional recovery (possibly because it exceeded the balance necessary for functional improvements), optional treatments with lower intensity or less time of WBV-therapy should be tested.

Slow axonal regrowth but extreme hyperinnervation of target muscle after suture of the facial nerve in aged rats.

Unilateral transection and suture of the facial nerve was performed in 60 old rats (20 months of age). The time course of mimetic reinnervation was studied by counting all retrogradely labeled motoneurons in the facial nucleus after injection of HRP into the whiskerpad muscles for 14-112 days post operation. The comparison between these neuron counts and data for young rats yielded four conclusions. First, the qualitative equivalent of the phenomenon "misdirected reinnervation" in aged rats was the same as in young adults: HRP-labeled motoneurons were scattered throughout the facial nucleus lacking myotopic organization from 18 until 112 days post operation. Second, no age-related loss of motoneurons was detected. Third, the axonal regrowth was delayed in aged rats. Fourth, the postoperative hyperinnervation (the projection of more motoneurons into a muscle than under normal conditions, i.e., the quantitative aspect of misdirected reinnervation) was more than two times higher than in young rats. These data may provide reasonable explanations for the poor functional recovery after reconstructive surgery on the facial nerve in old patients.

Recovery of original nerve supply after hypoglossal-facial anastomosis causes permanent motor hyperinnervation of the whisker-pad muscles in the rat.

Hypoglossal-facial anastomosis (HFA), used in humans for the treatment of facial palsy, was experimentally performed in adult female Wistar rats. The time course of facial reinnervation and the extent of the new motor nerve supply of the vibrissal muscles that develops after HFA were estimated by counting all motoneurons in the brainstem labeled by injection of horseradish peroxidase (HRP) into the whisker pad; muscle innervation by motor endplates was not studied. In untreated animals, HRP injection labels 1,254 +/- 54 (mean +/- S.D.; n = 6) motoneurons, localized exclusively in the lateral subdivision of the facial nucleus. Immediately following HFA, this number drops to zero. The first HRP-labeled motoneurons appear in the hypoglossal nucleus at 28 days postoperation (dpo) and at 56 dpo their number reaches 1,096 +/- 48. Unexpectedly, the facial nerve, whose proximal stump has been left as blind end during surgery, additionally sends axons to the facial periphery. This resprouting is first detected at 42 dpo with HRP-marked neurons throughout the facial nucleus lacking somatotopic organization. The number of these labeled neurons also rises with time, and at 56 dpo, a total of 1,797 +/- 142 facial and hypoglossal motoneurons, that is, 43% more motoneurons than in normal animals, supplies the whisker pad. This hyperinnervation, that is, the projection of more motoneurons into the target muscle than under normal conditions--further increases to 1,978 +/- 92 motoneurons at 224 dpo and may provide a new animal model for studying the competitive relationships between motoneurons in their search for peripheral targets.

Delayed hypoglossal-facial nerve suture after predegeneration of the peripheral facial nerve stump improves the innervation of mimetic musculature by hypoglossal motoneurons.

Surgical reconstruction of the facial nerve is common clinical practice following destruction of the intracranial facial nerve. Delayed hypoglossal-facial anastomosis (HFA) is the procedure of choice, although the effect of delay on outcome remains unclear. To study the effect of delayed anastomosis on reinnervation, we sutured the proximal stump of a freshly transected hypoglossal nerve of Wistar rats to the distal stump of the ipsilateral facial nerve, which had been transected 7-56 days earlier. Animals that had received HFA without delay served as the control group. Forty days after HFA, horseradish peroxidase (HRP) was injected into the whisker pad; 2 days later, the animals were killed. Reinnervation was assessed by determining the proportion of labeled neuronal cell bodies in the brainstem. The control group had 68% reinnervation of these muscles by hypoglossal neurons and had 32% reinnervation by facial neurons. When the distal facial nerve had been allowed to degenerate for 7 days before HFA, reinnervation of the hypoglossal nerve decreased to 54%, and reinnervation by the facial nerve increased to 46%. However, after a delay of 10-56 days, the hypoglossal fraction increased and stabilized at 77%, and the facial motoneuron fraction decreased to 23%. The presence of new neuromuscular junctions was confirmed by HRP labeling of motor end plates in vivo and by electromyography. We conclude that, under the conditions of hypoglossal-facial crossed nerve suture, the predegeneration of the distal stump of a transected facial nerve enhances the reinnervation of facial muscles by hypoglossal axonal sprouts.

Hypoglossal and reticular interneurons involved in oro-facial coordination in the rat.

Chewing, swallowing, breathing, and vocalization in mammals require precise coordination of tongue movements with concomitant activities of the mimetic muscles. The neuroanatomic basis for this oro-facial coordination is not yet fully understood. After the stereotaxic microinjection of retrograde and anterograde neuronal tracers (biotin-dextran, Fluoro-Ruby, Fluoro-Emerald, and Fluoro-Gold) into the facial and hypoglossal nuclei of the rat, we report here a direct bilateral projection of hypoglossal internuclear interneurons onto facial motoneurons. We also confirm the existence of a small pool of neurons in the dorsal part of the brainstem reticular formation that project ipsilaterally to both facial and hypoglossal nuclei. For precise tracer injections, both motor nuclei were located and identified by the electrical antidromic activation of their constituent motoneurons. Injections of retrograde tracers into the facial nucleus consistently labeled neurons in the hypoglossal nucleus. These neurons prevalently lay in the ipsilateral side, were small in size, and, like classic intrinsic hypoglossal local-circuit interneurons, had several thin dendrites. Reverse experiments - injections of anterograde tracers into the hypoglossal nucleus - labeled fine varicose nerve fiber terminals in the facial nucleus. These fiber terminals were concentrated in the intermediate subdivision of the facial nucleus, with a strong ipsilateral prevalence. Double injections of different tracers into the facial and the hypoglossal nuclei revealed a small, but constant, number of double-labeled neurons located predominantly ipsilateral in the caudal brainstem reticular formation. Hypoglossal internuclear interneurons projecting to the facial nucleus, as well as those neurons of the parvocellular reticular formation that project to both facial and hypoglossal nuclei, could be involved in oro-facial coordination.

Exogenous antigen containing perivascular phagocytes induce a non-encephalitogenic extravasation of primed lymphocytes.

Recent evidence suggests that T-lymphocyte extravasation and CNS-parenchymal infiltration during autoimmune disease might be regulated by antigen-presenting (ED2(+)) cerebral/spinal perivascular phagocytes (CPP/SPP). Since the massive erythrocytic and leukocytic infiltrates in the CNS of rats with experimental allergic encephalomyelitis do not allow a precise differentiation between CPP/SPP and the invading cells in the Virchow-Robin space, we developed a new immune-response model whereby the extravasation of T-lymphocytes was not followed by other blood cells. Adult Lewis rats were sensitized to horseradish peroxidase (HRP). Subsequent intracerebroventricular (i.c.v.) injections of HRP and/or Fluoro-Emerald (FE) served to: (1) challenge the primed T-lymphocytes and (2) label the CPP/SPP for additional immunocytochemical analysis. We found that 24 h and 3 days after single, double, or triple antigen boosting T-lymphocytes (R73(+), W3/25(+), OX50(+)) entered the Virchow-Robin space but did not break through the astrocytic glia limitans. Instead they adhered to HRP-containing activated CPP/SPP (mabs OX-6(+), SILK6(+), CD40(+), CD80(+), CD86(+)). This selective contact was mediated neither by cell adhesion molecules (P-selectin, ICAM-1, VCAM-1), nor promoted by chemokine receptors (CCR1, CCR5) or chemokines (monocyte chemoattractant protein (MCP)-1, MIP-1alpha, MIP-1beta, RANTES). This non-inflammatory, but antigen-dependent lymphocyte extravasation provides optimal conditions to further study the CNS immune response.

Changes in eye blink responses following hypoglossal-facial anastomosis in the cat: evidence of adult mammal motoneuron unadaptability to new motor tasks.

Hypoglossal-facial anastomosis is used in humans to restore the activity of the mimic musculature following irrecoverable facial nerve lesions. As eyelid movement kinetics is very well known, we have used this experimental model in cats to follow the evolution of blink responses and the adaptability of hypoglossal motor pools to new motor tasks. Although the electromyographic activity of the orbicularis oculi muscle in response to corneal air puffs, flashes of light or electrical stimulation of the supraorbital nerve was not recovered in the seven months following this crossed anastomosis, reflex blinks were got back by the increased activity of the retractor bulbi and extraocular recti muscles. The lid of the anastomosed side oscillated in perfect synchronization with tongue movements during licking, while it was severely affected in its motor function during optokinetic stimulation because of the spontaneous appearance of tongue-related hypoglossal activity. Present results suggest that adult mammal motoneurons are unable to readapt their motor programs to the kinetic needs of new motor targets and that most of the functional recovery observed in the cat was achieved by the compensatory hyperactivity of motor systems not directly affected by the surgery.

The cerebral perivascular cells.

This monograph reviews the literature and presents experimental data on the intracerebral presentation of antigen(s) to the immune system as a consequence of neuronal cell death. "Which cells are the antigen presenting cells (APC) of the brain?" is the main question of this book. The immune surveillance of the CNS occurs through specialized resident cells, which present (auto)antigen(s) to the immune system and thus initiate an (auto)immune response. There are four established prerequisites necessary to identify resident APC of the brain. First, the APC must be capable to phagocytose dead neurons. Second, in order to be recognized by T lymphocytes, these neuronophages must express Major Histocompatibility Complex (MHC) cells II glycoproteins on their surface. Third, in order to present (auto)antigen, the MHC class II-positive neuronophages must also be able to contact T lymphocytes. Fourth, in order to exert a stimulatory effect on T lymphocytes, the APC should be able to produce the cytokine interleukin-1 beta (IL-128 Mb). Three main tools were used to identify and characterize the APC of the brain. First, a lesion model was employed that yields a slowly progressing neuronal cell loss without disruption of the blood-brain barrier. This model consisted of resection of 10 mm of the facial nerve, which caused a slowly occurring neuronal death so that one year after resection the amount of facial neurons was about 44% of the control value. Second, neuronophages were labeled in vivo in situ via phagocytosis of the permanent fluorescent marker Fluoro-Gold (FG) from decaying pre-loaded facial motoneurons. Third, the FG-labeled neuronophages were immunocytochemically characterized with the new method "immunoquenching of fluorescence". Sections of the brainstem containing FG-labeled, i.e. fluorescent, neuronophages were incubated with a variety of primary antibodies, followed by avidin-HRP and DAB-nickel as a dark brown reaction product for bright-field microscopy. In the fluorescent mode this DAB reaction product selectively quenches the fluorescence of all immunopositive cells, i.e. only those neuronophages that do not bind to the primary antibody remain fluorescent. Combining FG-labeling of neuronophages with immunoquenching, a population of small round fluorescent cells was discovered, localized in the immediate vicinity of the motoneurons long after the neuronofugal migration of microglia. As the fluorescence of these cells was not quenched after a triple immunostaining with anti-neuronal-specific enolase, anti-GFAP and OX-42 (quenching all fluorescence from neurons, astroglia, and microglia), they seem to represent a new, immunologically unidentified neuronophage. Following this triple immunostaining, a broad panel of antibodies was tested to stain, quench fluorescence, and thus immunotype these enigmatic phagocytes. Only the monoclonal antibody ED2, the classical marker for perivascular cells, specifically stained the small round neuronophages. Although the perivascular cells are in the vicinity of the basal lamina of the cerebral vasculature, they must not be confused with the pericytes, which are not able to perform phagocytosis. In contrast, the perivascular cells are macrophages-ED2 recognizes an established macrophage membrane antigen. In addition, after neuronal injury a subset of the perivascular cells starts to synthesize MHC class II glycoproteins and IL-1 beta. Hence this population of cells seems to possess the complete machinery required for antigen presentation: They are macrophages, upregulate MHC class II molecules and IL-1 beta, and due to their anatomical location, have access to circulating T lymphocytes. What was still lacking, however, was a direct proof of neuronophagia. Our experiments provided this proof. (ABSTRACT TRUNCATED)