Aluminum adjuvants of vaccines injected into the muscle: Normal fate, pathology and associated disease.

Aluminum oxyhydroxide (Alhydrogel(®)) is a nano-crystalline compound forming aggregates that has been introduced in vaccine for its immunologic adjuvant effect in 1926. It is the most commonly used adjuvant in human and veterinary vaccines but mechanisms by which it stimulates immune responses remain ill-defined. Although generally well tolerated on the short term, it has been suspected to occasionally cause delayed neurologic problems in susceptible individuals. In particular, the long-term persistence of aluminic granuloma also termed macrophagic myofasciitis is associated with chronic arthromyalgias and fatigue and cognitive dysfunction. Safety concerns largely depend on the long biopersistence time inherent to this adjuvant, which may be related to its quick withdrawal from the interstitial fluid by avid cellular uptake; and the capacity of adjuvant particles to migrate and slowly accumulate in lymphoid organs and the brain, a phenomenon documented in animal models and resulting from MCP1/CCL2-dependant translocation of adjuvant-loaded monocyte-lineage cells (Trojan horse phenomenon). These novel insights strongly suggest that serious re-evaluation of long-term aluminum adjuvant phamacokinetics and safety should be carried out.

Testicular biodistribution of 450 nm fluorescent latex particles after intramuscular injection in mice.

The significant expansion in the use of nanoparticles and submicron particles during the last 20 years has led to increasing concern about their potential toxicity to humans and particularly their impact on male fertility. Currently, an insufficient number of studies have focused on the testicular biodistribution of particles. The aim of our study was to assess the distribution of 450 nm fluorescent particles in mouse testes after intramuscular injection. To this end, testes were removed from 5 groups of 3 mice each at 1 h (H1), 4 days (D4), 21 days (D21), 45 days (D45) and 90 days (D90) after the injection of 7.28 × 109 particles in the tibialis anterior muscles of each mouse. We examined histological sections from these samples by epifluorescence microscopy and confocal microscopy and identified testicular biodistribution of a small number of particles in groups H1, D4, D21, D45 and D90. Using CD11b immunostaining, we showed that particles were not carried into the testis by macrophages. The intratesticular repartition of particles mainly followed testicular vascularization. Finally, we found some particles in seminiferous tubules but could not determine if the blood-testis barrier was crossed.

Assessing cardiovascular risk factors after coronary artery bypass surgery: value of an aggressive strategy including systematic follow-up.

INTRODUCTION: Coronary revascularization surgery is a palliative treatment modality which should not preclude efforts to treat atherosclerosis. AIM: To assess ongoing cardiovascular risk factors after coronary artery bypass surgery and develop a strategy to attenuate such factors. METHODS: 108 patients requiring a coronary artery bypass were included: 2 died soon after surgery and 6 were excluded for personal reasons. 100 patients were re-admitted into hospital 7 months after surgery for risk factor assessment. Eight months later, they were re-contacted by telephone (systematic follow-up) for a re-assessment. RESULTS: The population consisted of 77 men with an average age of 64+/-11 years. Prior to the operation, the known risk factors were: smoking 34%; HBP 61%; cholesterol 47%; diabetes 30%; obesity 25%. During their hospital stay six months after the procedure: 91% of the patients had at least one lipid metabolism abnormality. New-onset diabetes was diagnosed in 5%. Blood pressure was uncontrolled in 18% and 10% were still smoking. Patients tended to be putting on weight and 55% engaged in little or no physical activity. Systematic follow-up: lipid metabolism had normalized in 70% of the patients. Blood glucose levels were significantly lower. Blood pressure was uncontrolled in 9% and 4% were still smoking. Their weight had stabilized and 65% were engaging in moderate-to-strenuous physical activity. CONCLUSION: Inadequate attention is paid to risk factors after coronary artery bypass surgery. A short hospital stay including a cardiovascular evaluation and education about risk factors has a positive impact on the management of atherosclerosis in the medium term.

Two temporal stages of oligodendroglial response to excitotoxic lesion in the gray matter of the adult rat brain.

Excitotoxic lesions in the gray matter induce profuse demyelination of passage and afferent fibers in areas of neuronal loss, independent of Wallerian degeneration. The time course of this phenomenon, which extends over weeks after the excitotoxin injection, suggests that demyelination is not related only to a direct effect of the toxin. In order to define mechanisms at work, a parallel study of myelin and oligodendrocytes was carried out following kainate injections into the adult rat thalamus. Within the 1st day postlesion, myelin alteration appeared throughout the area exhibiting neuronal loss, while the number of oligodendrocytes fell by 45%. No apoptotic oligodendrocytes were identified at that time. Over the following 2 days, there was no further loss of myelin and oligodendrocytes, but there was an increase in the number of oligodendrocytes displaying typical signs of apoptosis as revealed with TUNEL-end-labeled nuclei, Hoechst-labeled condensed chromatin bodies, or bax immunoreactivity. This resulted in a second, progressive loss of both myelin and oligodendrocytes leading to their almost complete disappearance 2 weeks postlesion. These results demonstrate two temporal stages of oligodendroglial cell death. The excitotoxin injection resulted in the rapid destruction of a first oligodendroglial population, most probably by necrosis. A second population died in a delayed manner from apoptosis. This second wave of death coincided with an activated microglia/macrophage invasion of the lesion, suggesting that delayed oligodendroglial death results from toxic microglia/macrophage effects. In addition, the longest surviving oligodendrocytes were located next to reactive astrocytes, suggesting the existence of trophic interactions between these two glial populations.

Transforming growth factor alpha: a promoter of motoneuron survival of potential biological relevance.

Expression of transforming growth factor alpha (TGFalpha), a member of the epidermal growth factor (EGF) family, is a general response of adult murine motoneurons to genetic and experimental lesions, TGFalpha appearing as an inducer of astrogliosis in these situations. Here we address the possibility that TGFalpha expression is not specific to pathological situations but may participate to the embryonic development of motoneurons. mRNA of TGFalpha and its receptor, the EGF receptor (EGFR), were detected by ribonuclease protection assay in the ventral part of the cervical spinal cord from embryonic day 12 (E12) until adult ages. Reverse transcription-PCR amplification of their transcripts from immunopurified E15 motoneurons, associated with in situ double-immunohistological assays, identified embryonic motoneurons as cellular sources of the TGFalpha-EGFR couple. In vitro, TGFalpha promoted the survival of immunopurified E15 motoneurons in a dose-dependent manner, with a magnitude similar to BDNF neuroprotective effects at equivalent concentrations. In a transgenic mouse expressing a human TGFalpha transgene under the control of the metallothionein 1 promoter, axotomy of the facial nerve provoked significantly less degeneration in the relevant motor pool of 1-week-old mice than in wild-type animals. No protection was observed in neonates, when the transgene exhibits only weak expression levels in the brainstem. In conclusion, our results point to TGFalpha as a physiologically relevant candidate for a neurotrophic role on developing motoneurons. Its expression by the embryonic motoneurons, which also synthesize its receptor, suggests that this chemokine is endowed with the capability to promote motoneuron survival in an autocrine-paracrine manner.

Glial and endothelial cell response to a fetal transplant of purified neurons.

Astrocytes, microglia and endothelial cells display very specific phenotypic characteristics in the intact adult CNS, which appear quite versatile when grown in culture without neurons. Indirect evidence from in vitro co-culture studies and analysis of the effects of specific neuronal removal in vivo, does accordingly favour a role of neurons for the phenotypic repression of these cells in the intact brain. In order to provide more direct evidence for such neuronal influence, we attempted to induce, in the rat brain, a reversal of the post-lesional activation of astrocytes, microglia and endothelial cells by transplantation of fetal neurons purified by immunopanning. Host microglial cells which have been activated by the lesion process, penetrated the neuronal graft during the few days after the transplantation. Reactive astrocytes began to appear in the lesioned parenchyma and gathered around the transplant. Thereafter they first sent their processes in the direction of the neuronal graft, before they migrated into the graft a few days later. At this time, which was at the end of the first week post-transplantation, the host endothelial cells sprouted "streamers" of basal lamina within the graft forming small capillaries. During the second week post-transplantation, numerous astrocytes and microglial cells, both displaying a reactive hypertrophied morphology, were observed throughout the grafts. Finally, by the end of the first month, the activated cells differentiated towards a quiescent, resting morphology. At this time the grafts contained a vascular network with morphological characteristics comparable to those observed in the intact brain parenchyma. The results indicate that the interaction of activated astroglia and microglia and endothelial cells with neurons causes the cells to re-differentiate and regain phenotypic features characteristic of intact brain parenchyma, strongly suggesting that neurons play an essential role in the phenotypic restriction of glial and endothelial cells in the adult central nervous system.

Long-term histological follow-up of genetically modified myoblasts grafted into the brain.

Although primary muscle cells have been used as intracerebral vehicles for transgene expression in the past, data concerning their long-term survival after grafting into the brain, and the reaction of the host tissue to their implantation are lacking. In order to study these aspects, we have implanted, into the brain, primary muscle cells infected ex vivo with recombinant retroviruses carrying the E. coli LacZ gene. The muscle cells were delivered stereotaxically into different areas of the brain of adult rats and the grafts were analyzed up to 105 days after implantation. Intraventricular implantations did not lead to surviving grafts. In contrast, myoblasts developed when they were grafted into gray or white matter regions. They appeared numerous during the first weeks, but decreased dramatically in number over time. Over months, the grafts appeared to fill up with collagen. Astrocytes elaborated a continuous glia limitans surrounding the implant. Blood vessels coming from the host tissue were found within the grafts. The blood-brain barrier was permanently disrupted within the transplants. beta-Galactosidase activity was abundant during the first weeks, but decreased to a very low level subsequently. This decrease paralleled that of the number of muscle cells. In conclusion, myoblasts transplanted into the adult brain survived only temporarily, which implies a transient transgene expression. In addition, before being eliminated, muscle cells were surrounded by a glia limitans, which may limit exchanges with the host tissue. Altogether, these results suggest that intracerebral transplantation of myoblasts may possibly provide a relevant vehicle only for short-term delivery of a gene product.

Morphological maturation of thalamic neurons as studied in fetal neural transplants.

This study attempts to determine whether fetal thalamic neuroblasts from rat embryos (embryonic age 15 days) labeled with horseradish peroxidase (HRP) can differentiate into their normal dendritic phenotype when transplanted as a cell suspension into a lesioned site in the adult somatosensory thalamus. The HRP labeling provided a Golgi-like staining of numerous neurons up to 12-14 days after transplantation. There were three main results. 1) As early as 2 days after transplantation three morphologic cell types were observed: Two were bipolar and the third multipolar. These cellular profiles are characteristic of adult ventroposterolateral, reticular, and ventroposteromedial neurons and suggest that transplanted neurons can take shape in the absence of specific arrangements of afferent fibers. 2) The initial stage of dendritic growth was characterized by numerous growing specializations and consisted of a rapid, arborizing growth that appeared to proceed at an accelerated rate relative to normal development. During the later stage, which was characterized by the great reduction of growing specializations, dendritic remodeling resulted in a simpler morphology, and the transplanted neurons did not achieve an adult morphology. 3) Putative axons exhibiting growth cones were present in impressive densities in the transplants, and a number of them grew into the neuron-depleted host thalamus. A very small number of axons grew into host gray matter outside the lesioned area, indicating that neurodegenerative areas provide a better substrate for neurite outgrowth than intact tissue. In rare instances axons were visible in the internal capsule, indicating that the biochemical inhibition provided by mature myelin and oligodendrocytes may not be an absolute obstacle to axonal growth.

Transforming growth factor alpha (TGF alpha) expression in degenerating motoneurons of the murine mutant wobbler: a neuronal signal for astrogliosis?

The enhanced expression of the trophic factor transforming growth factor alpha (TGF alpha) in reactive astrocytes following CNS injury suggests that TGF alpha has a role in the development of astrogliosis. We explored this hypothesis in the murine mutant wobbler, which presents a progressive motoneuronal degeneration associated with an astrogliosis. Evolution of astrogliosis, and expression of TGF alpha precursor (pro-TGF alpha) and of its receptor were examined over the course of the disease, using genetically diagnosed animals and immunocytochemical techniques. We report here that degenerating motoneurons of the cervical spinal cord and a subset of astrocytes express pro-TGF alpha, prior to the onset of astrogliosis, when the first clinical manifestations of the disease are observed at 4 weeks of age. TGF alpha expression appeared strongly correlated with motoneuronal degeneration. All pro-TGF alpha-immunoreactive neurons exhibited a degenerative morphology, and the number of pro-TGF alpha-immunoreactive neurons increased with the progression of the disease. At the glial level, we observed that astrogliosis was a transitory phenomenon in the wobbler mice, developing in coordination with the motoneuronal expression of pro-TGF alpha. Astrogliosis became evident in 6-week-old wobbler mice, when the number of pro-TGF alpha-immunoreactive motoneurons was maximal, and regressed in older mutant mice in correlation with the disappearance of pro-TGF alpha-immunoreactive motoneurons. Furthermore, TGF alpha/EGF receptor immunoreactivity was exclusively localized in a subset of reactive astrocytes, its expression following closely the course of the astrogliosis. These data show that TGF alpha synthesis by the affected motoneurons is an early event in the course of the wobbler disease, and suggest a role for TGF alpha as a neuronal inducer of astrocytic reactivity.

In vivo transfer of a marker gene to study motoneuronal development.

Adenovirus vectors containing a marker gene (lacZ from Escherichia coli) are potent for transferring the gene to neurones after intraparenchymal injections. Expression of the marker gene may lead to the synthesis of an enormous amount of beta-galactosidase which diffuses throughout the entire neurone, providing a 'Golgi-like' staining. This suggested that the technique may be used to study the morphology of specific neuronal populations. We have validated this hypothesis by analysing the postnatal development of motoneurones in the rat cervical cord. Injections of the viral suspension into one ventral horn were performed at different ages after birth. Histochemical staining using X-Gal revealed morphological changes occurring within the first 3 weeks with enlargement of the perikaryon and increased dendritic complexity. Immunoreactivity for CGRP was visualized in double-staining experiments. In vivo transfer of a marker gene therefore provides a new way to analyse neuronal morphology which allows selection of the cells to be studied and double-labelling with immunohistochemical markers.

Maturation and integration of purified foetal neurons transplanted into the adult brain.

Fetal neural transplants presently developed as a therapeutic strategy for neurodegenerative diseases include both the neurons of interest and their cellular environment. These glial and vascular cells may be detrimental by, for instance, expressing foreign MHC antigens. This study was undertaken to determine whether purified neurons would survive transplantation into an adult host brain. Embryonic rat spinal neurons were purified by panning and transplanted into adult hosts' brain. During the first three weeks post-transplantation the grafts contained essentially packed immature neurons. Later transplants contained large, multipolar neurons, demonstrating the ability of transplanted neurons to mature in the adult environment. The adult host appears actively involved in the integration of such a transplant by complementing it with microglial and vascular cells.

[Connections of the posterior region of the thalamus in rats]. / Etude des connexions de la région postérieure du thalamus chez le rat.

Using retrograde and anterograde tracing methods we have studied in the posterior region of the thalamus of the rat the distribution of: (1) the terminal fields of the main afferents arising from somatosensory centers (dorsal column nuclei, interpolar trigeminal subnucleus, somatosensory cortex), motor centers (red nucleus, motor cortex) and multimodal structures (deep layers of the superior colliculus, zona incerta, cingular cortex) and of (2) the neurons giving rise to the main efferents towards the sensorimotor cortex, the red nucleus, the deep layers of the superior colliculus and the zone incerta. The overlap of the retrograde and anterograde labeling reveals a relatively homogeneous region. Considering however the cortical connections, three different subdivisions can be distinguished: a caudal pole completely devoid of cortical connections, a medial subdivision receiving cortical afferents from the sensorimotor and cingulate cortices and a rostral pole reciprocally connected with the sensorimotor cortex. Therefore the rostral pole would be the only part of this region which should be included in the thalamus.

Cortical and subcortical connections of the pars compacta of the anterior pretectal nucleus in the rat.

The efferent and afferent connections of the dorsal part of the anterior pretectal nucleus, pars compacta (APc), were studied experimentally in the rat by using neurotracers. A restricted number of structures supply afferents to the anterior pretectal nucleus: the visual cortex (areas 17, 18 and 18a), ventral lateral geniculate nucleus and superficial layers of the superior colliculus. Additional afferents have been demonstrated originating from the Darkschewitsch nucleus, periaqueductal gray, zona incerta and anterior cingulate cortex. Efferent fibers are distributed to a sector of the deep mesencephalic nucleus just dorsolateral to the red nucleus, the basilar pontine gray, posterior and olivary pretectal nuclei, superficial layers of the superior colliculus, lateral posterior thalamic nucleus, ventral lateral geniculate nucleus and zona incerta. These anatomical observations indicate that the pars compacta of the anterior pretectal nucleus is closely related to visual centers, suggesting an involvement of this nucleus in visually mediated behavior.

Fetal neural transplants into an area of neurodegeneration in the spinal cord of the adult rat.

Lesioning the spinal cord with an excitotoxic agent provides a model of neuronal degeneration while sparing afferent axons. The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension were able to rebuild a neural circuitry in the neuron-depleted adult cord. Fetal spinal cords, taken from rat embryos (gestational day E12-13), were transplanted as cell suspensions into an area of the lumbar cord previously depleted of neurons using kainic acid. The excitotoxic lesion extended over ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb. During the first month after injection, the damaged cord was characterized by proliferation and recruitment of various glial cell and Schwann cell populations. First to appear were activated microglia/macrophages and next reactive astrocytes which entered the lesion from its borders with the intact tissue. Schwann cells also ensheathed central axons. Differential sensitivity of various afferents to loss of postsynaptic target neurons was observed: rubrospinal and corticospinal afferents decreased in density while no conspicuous changes were observed for immunostained CGRP-containing or monoaminergic fibers. Two to fourteen months after surgery, transplants occupied most of the neuron-depleted area. The grafts did not display a laminar organization. Monoaminergic afferents grew for a long distance and formed a network within transplants. Similarly, primary sensory CGRP-immunoreactive fibers entering in the dorsal roots penetrated deeply into transplants. In contrast, cortico- and rubrospinal afferents entered only the most peripheral portion of transplants. Our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord. Host-to-graft connections can be formed, although their spatial extent in the transplants may depend upon features of the afferent fiber systems.

Skilled forelimb use in the rat: amelioration of functional deficits resulting from neonatal damage to the frontal cortex by neonatal transplantation of fetal cortical tissue.

Limb preference and dexterity of right and left forelimbs were studied in a food reaching task in normal rats (control group), in rats that sustained a neonatal lesion of the left frontal cortex (lesion group) and in animals that received a transplant obtained from the frontal cortex of 16-day-old embryos immediately after the lesion (graft group). In addition, an anatomical study of host-transplant interconnections was performed in several transplanted animals. The results indicate that the lesion slightly increased the preference for the limb ipsilateral to the lesion and transplantation of fetal cortical tissue did not restore the preference for the contralateral limb. Furthermore, lesion of the motor cortex induced a deficit in the dexterity of limb use in the food-reaching task. This motor deficit was more pronounced when the limb contralateral to the lesion was used. Transplantation of embryonic cortical tissue led to a reduction of the motor deficit. Compared to the lesion group, the graft group had a higher success rate and a lower percentage of motor abnormalities, whichever forelimb was used, ipsi- or contralateral to the transplant. Nevertheless, larger improvement was noted with contralateral forelimb usage. Functional recovery was not complete since the control group still performed significantly better than the graft group, although almost complete sparing in skilled reaching was noted when the limb ipsilateral to the transplant was used. Analysis of host-transplant interconnections indicates that the transplants sent fibers to the host spinal cord, caudate-putamen, thalamus and homotopic contralateral cortex and received projections from the host thalamus and contralateral cortex. It is therefore suggested that neonatal transplantation of fetal cortical tissue promotes functional recovery from damage to the motor cortex occurring at birth.

Fetal cortical transplants reduce motor deficits resulting from neonatal damage to the rat's frontal cortex.

Motor deficits in the execution of grasping movements of the right forelimb were compared in normal female Wistar rats, in animals which sustained a neonatal lesion of the left frontal cortex and in animals which received immediately after the lesion a transplant obtained from the frontal cortex of E16 embryos. Behavioral testing was carried out from postnatal day 48 (D48) to D108. The animals were placed at the center of a circular wire grid which was turned upside down so that they hung by their 4 paws at a distance of 40 cm above the floor. The precision of grasping movements of the right limb and the number of falls were recorded during a 1 min session of active moving across the grid. The lesioned subjects were most impaired on both motor indices whereas the grafted animals although performing slightly poorer than the controls were, however, less impaired than the lesioned animals. Fetal cortical transplants, therefore, seem to promote functional recovery from neonatal cortical damage.

Distribution of the efferent projections of the rat posterior thalamic nucleus as revealed by Phaseolus vulgaris immunohistochemistry.

Following iontophoretic injection of Phaseolus vulgaris in the rat posterior thalamic nucleus (PT) dense terminal fields were labeled in the ipsilateral red nucleus, zona incerta, intermediate and deep layers of the superior colliculus, pretectum, basilar pontine gray, and dorsal pole of the caudal division of the ventral lateral thalamic nucleus. Several other structures were more sparsely labeled; the reticular gigantocellular and pontine oral nuclei, nucleus of the mesencephalic tract of the trigeminal nerve, deep mesencephalic and posterior hypothalamic nuclei and the rostral part of the lateral posterior thalamic nucleus. The motor and, to a lesser extent, somatosensory cortices were labeled only following tracer injection in the rostral PT. The posterior thalamic nucleus thus projects to several centers primarily treating somatosensory and/or nociceptive messages, and to precerebellar and motor nuclei. Since a previous study has established that the PT also receives a major somatosensory and/or nociceptive input (Roger and Cadusseau, 1984), a non-specific, modulatory role of the PT is therefore proposed in the treatment of some aspects of somatosensory and/or nociceptive information.

Direct neuronal and macroglial versus indirect macrophagic labeling in transplants of fetal neural tissue incubated with gold particles.

In a recent light microscopic study based on the transplantation of fetal cells previously incubated with gold-loaded Sendai viruses, S. C. Ardizzoni, A. Michaels, and G. W. Arendash (1988, Science 239: 635-637) proposed that fetal neurons could migrate out into an adult host brain. This result was puzzling in view of several previous contradictory studies, and the light microscopic identification of labeled cells as neurons could be questioned. The present study was therefore undertaken to replicate this study but using electron microscopy to definitely identify migrating gold-labeled elements. Analysis was performed in a model of thalamic transplantation in which previous labeling studies, using thymidine or horseradish peroxidase, had failed to demonstrate any neuronal migration. Gold particles combined with wheat germ agglutinin peroxidase were used to label the fetal tissue prior to transplantation. Fetal cells did incorporate the protein gold complex, and the gold particles were visualized in transplants up to at least 2.5 months after grafting. Electron microscopy analysis reveals that the gold particles are internalized and retained in the lysosomes of grafted neurons and glial cells. In no transplant could labeled grafted neurons be observed intermingled with host neurons, outside the limits of the transplant. In contrast, heavily labeled macrophages were observed surrounding the transplants and sometimes migrating away from the transplant-host interface into the host tissue. These macrophages, presumably of host origin, can be recognized at the light microscopic level by their yellowish staining, a cellular characteristic already noted by Ardizzoni et al. for gold-labeled migrating elements.

Description of a large projection from the mesodiencephalic junction to the rostral red nucleus. An anatomical study in the rat and in the cat.

Horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) were deposited in the rostral pole (Rpc) of the red nucleus in 6 rats and 5 cats. In the rat, a zone of dense retrograde cell labeling and of diffuse axonal labeling occurred in the sub-pretectal area, a region which corresponds to the posterior thalamic nucleus (PT) of Bold et al. (Brain Research, 12 (1984) 521-527). In the cat, a similar retrograde and anterograde pattern of labeling was observed. The labeled area extended from the deep pretectum up to the diencephalon above the dorsomedial aspect of the ventral posterior thalamic nucleus. In a second set of experiments, 14 rats received a tracer injection (HRP, WGA-HRP or Phaseolus vulgaris-leucoagglutinin) in the sub-pretectal area. The resultant pattern of labeling consisted in a heavy anterograde terminal labeling within the Rpc of the red nucleus. Sparse retrograde cell labeling also occurred in the Rpc.