[MEDCARE for People in Eritrea : A report on 9 years' help through self-help]. / MEDCARE for People in Eritrea : Ein Bericht über 9 Jahre Hilfe zur Selbsthilfe.

The current article is an experience report on the establishment of an ENT clinic in Asmara/Eritrea and the organization of regular work stays for the further education of local colleagues. Objectives of the project are content and structural support for self-help and thus achievement of sustainable development aid, which benefits both the medical development of the country and the care of the local patients.

Axotomy induces transient calbindin D28K immunoreactivity in hypoglossal motoneurons in vivo.

Calbindin D28K, an intracellular calcium-binding protein, acts as Ca2+ buffering system in the cytoplasm. By means of this property, calbindin may protect neurons against large fluctuations in free intracellular Ca2+ and, hence, may prevent cell death. Although axotomy causes a massive influx of calcium into the lesioned neurons, resection of the hypoglossal nerve does not induce extensive neuronal cell death in rats. Even several weeks after axotomy, about 70% of the motoneurons survive despite permanent target deprivation. The mechanisms responsible for this remarkable survival rate are unknown. In this study, we have looked at the modification of calbindin immunoreactivity in axotomized hypoglossal motoneurons. In non-axotomized motoneurons, no calbindin is detectable by immunocytochemistry. Axotomy induced an increase of calbindin immunoreactivity in lesioned motoneurons. This increase, visualised by the number of calbindin-immunoreactive neurons extended from 1 day to 28 days. At this time most, but not all, motoneurons located on the side of the lesion were calbindin-positive as shown by retrograde labeling and immunoquenching. From 14 days post operation, calbindin immunoreactivity decreased and reached its basal value after 35 days post operation. At that time, only fibres were still calbindin immunoreactive. Interestingly, calbindin-immunoreactivity was also increased in almost all cell nuclei, compatible with a nuclear regulation. These data are consistent with the hypothesis that, as a reaction to axotomy, motoneurons trigger an increase in calbindin expression which acts as a compensatory Ca(2+)-buffering system, enabling neurons to maintain Ca2+ homeostasis and the survival of many motoneurons after axotomy.

Expression of different isoforms of nitric oxide synthase in experimentally denervated and reinnervated skeletal muscle.

Denervated muscle fibers express enhanced levels of stress and apoptosis-associated proteins and undergo apoptosis. In experimentally denervated and reinnervated rat facial muscle, we now evaluate changes in the expression patterns of different isoforms of nitric oxide synthase (NOS)-generating nitric oxide (NO), which mediates oxidative stress and apoptosis. Physiological expression of NOS corresponds to a constant sarcolemmal staining pattern for neuronal NOS (nNOS) and a patchy sarcolemmal and weak sarcoplasmic labeling for the endothelial NOS-isoform, with no expression for inducible NOS (iNOS). Denervated muscle displayed distinct downregulation of nNOS with preserved expression of dystrophin. Also, denervated and immediately reinnervated muscle fibers showed decreased expression of nNOS. However, muscle fibers reinnervated for 10 weeks revealed a restored physiological expression of nNOS. There were no changes in the expression of endothelial and inducible NOS. As NO is known to induce growth arrest and collapse of neuronal growth cones, downregulation of NOS may contribute to promotion of axonal regeneration by aiding formation of new endplates. NO is upregulated in reinnervated muscle fibers and thus prevents polyneural hyperinnervation by extrajunctional synapses. Furthermore, downregulation of NOS during denervation is compatible with the finding that low levels of NO contribute to apoptosis instead of necrosis in disease states of oxidative stress.

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.

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.

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.

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.

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.

Accelerated split-course radiation and simultaneous chemotherapy in patients with unresectable head and neck-cancer.

Forty-nine patients with unresectable squamous cell carcinomas of the head and neck were treated with accelerated radiotherapy (2 x 2.1 Gy/day, day 1-4 in week 1,2,5 and 6, total dose of 67.2 Gy) and simultaneous carboplatin (50 Mg/M2/ treatment day). Mucositis (21% grade 3 and 4, WHO) and leukopenia (40% grade 3 and 8% grade 4, WHO) were the most important side effects but did not limit the treatment schedule. The response rate was: 46.5% CR (20 pts), 46.5% PR (20 pts), 5% NC (2 pts) and 2% PD (1 pt). After three years overall survival was 35% (median 14 month) and in complete responders disease-free survival was 52%. Our results indicate that combined accelerated radio-chemotherapy might improve the poor results achieved with conventional radiotherapy or sequential chemo-radiotherapy in this difficult patient population. Further studies are neccessary to clarify whether modified radiotherapy or simultaneous chemotherapy or the combination of both are the reason for the improved treatment results.

Induction chemotherapy in primary resectable head and neck tumors - a prospective randomized trial.

Although induction chemotherapy prior to local therapy produces encouraging initial response rates in head and neck cancer, randomized studies have failed to demonstrate an advantage in survival. All randomized studies included only patients with far advanced stage III and IV disease which appears to be the main reason for the low rate of complete responses (max. 18%) in these trials. According to statistical considerations nearly 50% complete responders are necessary before improved survival benefit can be expected. Until now, such complete response rates are only achieved with induction chemotherapy in resectable T2-T3, N0-N2 disease. Therefore, we started a prospective randomized trial including only patients with these stages of disease. Patients were stratified by primary tumor site and neck node status and were randomized to receive either induction chemotherapy with three cycles of carboplatin/5-FU prior to surgery and radiotherapy (arm A, 49 patients) or standard treatment with surgery and radiotherapy (arm B, 47 patients). Patients were stratified by primary tumor site and neck disease. After a follow-up of 12-48 months overall survival was 72% in arm A and 53% in arm B (n.s.). Considering only the patients with cancer of the oral cavity and the tonsil overall survival was 88% in arm A and 44% in arm B (p<0.05). As of today, the number of patients with carcinomas of the hypopharynx and base of tongue is too small for a statistically significant statement, but preliminary data indicate a better overall and disease-free survival without chemotherapy in these patients. Therefore, we recommend controlled trials with induction chemotherapy in patients with primary resectable carcinomas of the oral cavity and the tonsil, stages T2-T3 and N0-N2, prior to surgery but not in patients with cancer of the hypopharynx and base of the tongue.

Nimodipine-accelerated hypoglossal sprouting prevents the postoperative hyperinnervation of target muscles after hypo glossal-facial anastomosis in the rat.

Hypoglossal-facial anastomosis (HFA), used for the treatment of facial palsy, was performed in adult Wistar rats. For 7-224 days post operation (DPO), half of the animals were kept on standard laboratory food and half received food pellets containing 1000 ppm of the Ca2+ channel blocker nimodipine. The postoperative neurotization of facial muscles in these two groups was traced by comparing numbers of all retrogradely labeled neurons after injection of HRP into the whiskerpad muscles. In unoperated animals, injection of HRP labeled 1254 ± 54 neurons. Immediately after HFA, this number dropped to zero. The treatment with nimodipine yielded two beneficial effects. (1) In the early phase of regeneration (until 28 DPO), it accelerated the sprouting of hypoglossal axons into the facial periphery; (2) In the final phase, it suppressed the axonal sprouting from both, hypoglossal and facial stumps. In this way nimodipine fully prevented the postoperative hyperinnervation, i.e. the projection of more hypoglossal plus facial motoneurons to the whiskerpad muscles than under normal conditions.

The use of texture analysis to study the time course of chromatolysis.

Image analysis of the textural feature entropy of the Nissl substance was used to monitor the time course of chromatolysis in regenerating hypoglossal motoneurons and degenerating facial motoneurons 4-112 days after hypoglossal-facial anastomosis in rats. Changes in the Nissl substance were detected that were not obvious on the basis of subjective judgement of the light-microscopical appearance of the neurons. Chromatolysis started 4 days post operation (dpo) and was not reversed at 112 dpo in both nuclei. The increase of chromatolysis was 14-28 dpo faster in the regenerating hypoglossal neurons than in degenerating facial neurons. Maximal chromatolysis was measured at 56-70 dpo in both nuclei. Afterwards chromatolysis persisted at a significantly higher level in the degenerating facial motoneuron pool. In conclusion, chromatolysis is a very long persisting reaction. In the beginning chromatolysis is faster and greater in regenerating rather than in degenerating neurons. In contrast, passing the maximal reaction, chromatolysis is maintained at a higher level in degenerating motoneurons. Image analysis of textural features is a suitable and reliable tool to monitor the time course of neuronal cell body changes. The presented quantitative method could be applied in any neurobiological study influencing the regeneration or degeneration of motoneurons.

Nitric oxide synthase in the vestibulocochlear system of mice.

The exact distribution of nitric oxide-synthases (NOS) and the NO-target enzyme soluble guanylyl cyclase (sGC) in the cochlea and vestibular organ is an issue of current discussion. The existence of NOS-isoforms in the cochlea of the guinea pig has been described recently, while information about the vestibular system are still rare and non-satisfying. In order to gain more information, immunostaining was performed, using specific antibodies to NOS I-III and to sGC, on paraffin sections of complete temporal bones from mice. NOS III could be detected in cochlea and vestibular ganglion cells, in nerve fibres, in outer hair cells of the cochlear and in the sensory epithelium of the maculae. Also, the spiral ligament and the limbus epithelium was positive to NOS III. NOS I was found in the sensory epithelium of the maculae and cristae ampullares, outer and inner hair cells of the cochlea, in nerve fibres and in ganglion cells. In contrast to that NOS II could not be detected at all. Furthermore, a strong NOS I immunoreaction was displayed on the endosteum of the bone, while the periosteum was lacking of NOS. NOS detection was accompanied by immunoreactivity to sGC. The findings imply that NOS I and III-generated NO is involved in neurotransmission and other regulative processes in the vestibulocochlear system.

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the cochlea of guinea pigs after intratympanical endotoxin-treatment.

Since NO is believed to be involved in cochlear physiology, presence of the constitutive isoforms of nitric oxide synthase (NOS), and the target enzyme of NO, soluble guanylyl cyclase (sGC) in structures of the mammalian cochlea have been demonstrated. To date, no reports have been published regarding the detection of the inducible isoform (NOS II) in the cochlea. In order to show the capability of iNOS expression in cochlear tissue, a mixture of proinflammatory bacterial lipopolysaccharides (LPS) and tumor necrosis factor alpha (TNF-alpha) was injected into the tympanic cavity of guinea pigs, vs. saline-solution as control. Paraffin sections of LPS/TNF-alpha treated and saline-treated cochleae (6 h) were examined immunohistochemically with specific antibodies to neuronal, endothelial and inducible NOS and to sGC. Initiated expression of iNOS in the cochlea was observed in the wall of blood vessels of the spiral ligament (SL) and the modiolus, in supporting cells of the organ of Corti, in the limbus, in nerve fibers and in a part of the perikarya of the spiral ganglion after LPS/TNFalpha-treatment. iNOS was not detected in saline-treated control tissue. Expression of both constitutive NOS-isoforms (endothelial and neuronal NOS) and of sGC showed no significant differences in both experimental groups. Endothelial eNOS and neuronal bNOS were detected co-localized in ganglion cells, in nerve fibers, in cells of the SL and in supporting cells of the organ of Corti, but not in sensory cells. Strong labeling for bNOS became evident in the endosteum of the cochlea, while in the endothelium of blood vessels and in the epithelium of the limbus only eNOS could be labeled. sGC could be detected in SL, in supporting and sensory cells of the organ of Corti, in nerve fibers, ganglion cells, in the wall of blood vessels and in the limbus-epithelium. While small amounts of NO, generated by bNOS and eNOS, seem to support the cochlear blood flow and auditory function as well as neurotransmission, high amounts of iNOS-generated NO could have dysregulative and neurotoxic effects on the inner ear during bacterial and viral infections of the middle and inner ear.

Localisation of the nitric oxide (NO)/cGMP-pathway in the vestibular system of guinea pigs.

The exact distribution of nitric oxide-synthases (NOS) in the vestibular system has not been described satisfying yet. Immunostaining, using specific antibodies to the three known NOS-isoforms, to cyclic guanosine monophosphate (cGMP) and soluble guanylyl-cyclase (sGC), the second messenger system of nitric oxide (NO), was performed on paraffin sections of temporal bone from guinea pigs. eNOS could be detected in vestibular ganglion cells and in nerve fibres, including the calyces, surrounding the type 1 hair cells (HC). bNOS was found in the sensory epithelium, ganglion cells and in bone, while iNOS could not be found. NOS-detection was accompanied by reactivity to sGC and to cGMP. This finding implies that b- and eNOS-generated NO is involved in regulative processes in neurotransmission and regulation of blood flow.

Expression of inducible nitric oxide-synthase in the vestibular system of hydropic guinea pigs.

Immunohistochemical investigations of the guinea pig vestibular system, using a specific antibody to the inducible isoform of NO-synthase (iNOS/NOS II), have been performed 3 weeks after surgical closure of the right endolymphatic duct (n = 7). Endolymphatic hydrops (ELH) of the right temporal bone became evident by excavation of the Reissner's membrane in all seven animals. Those animals revealed iNOS-expression in ganglion cells, in the wall of blood vessels and in nerve fibers of the right vestibular system, while the corresponding left temporal bones and temporal bones of non-operated controls (n = 6) as well as of sham-operated animals (n = 3) did not show any iNOS-positive structures. iNOS-generated NO could be involved in the pathophysiology of vestibular dysfunction in Meniere's disease.

Detection of extracellular signal-regulated kinase1/2 in the inner ear of guinea pigs.

Growth factors, such as vascular endothelial growth factor (VEGF) and neurotrophins, recently identified in the inner ear of guinea pigs, exert their proliferative properties partly through activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). In order to demonstrate presence of ERK1/2 in the inner ear we performed immunohistochemical analysis using specific antibodies to inactive and activated ERK1/2 on paraffin-sections of temporal bones from guinea pigs (n=5). In the cochlea clear immunoreactivity to inactive ERK1/2 was predominant in the spiral ligament, in the organ of Corti (intensive staining in supporting cells, faint staining in sensory cells) and limbus epithelium, while spiral ganglion cells and nerve fibres revealed weak staining. Activated ERK1/2 could be detected sparely in the spiral ligament exclusively. In the vestibule inactive ERK1/2 was located in the sensory epithelium, in nerve fibres and in vascular endothelium, while activated ERK1/2 could be detected in few nerve fibres and synaptic endings (buttons and calyces) on hair cells of the maculae and crests and in the endothelium of few blood vessels. These findings provide evidence that activated ERK1/2, as a general downstream signal of growth factors, may be contributed in the inner ear physiology.

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the hydropic cochlea of guinea pigs.

Immunohistochemical investigations of the guinea pig cochlea, using a specific antibody to the inducible isoform of NO synthase (iNOS/NOS II), have been performed 3 weeks after closure of the right endolymphatic duct (n=7). Endolymphatic hydrops, the morphological substrate of Meniere's disease, became evident by distension of the Reissner's membrane. iNOS expression could be noted in endothelium, spiral ganglion cells, in nerve fibers, in supporting cells of the organ of Corti and cells of the spiral ligament. Temporal bones of non-operated controls (n=6) as well as of sham-operated animals (n=3) did not show structures positive to iNOS. These findings imply that iNOS-generated NO could be involved in the pathophysiology of cochlear dysfunction in Meniere's disease.

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.

Changes of the compound action potential (CAP) and the expression of inducible nitric oxide synthase (iNOS/NOS II) in the cochlea under the inflammatory condition(1).

In this study, the effect of endotoxin on the guinea pig cochlea has been examined electrophysiologically and immunohistochemically. Bacterial lipopolysaccharide (LPS, 5 mg/ml, 0.2 ml) was injected into the middle ear trans-tympanically. The electrocochleograms were measured before, immediately upon, and 3, 6 and 12 h after the injection continuously with an electrode inserted into the facial canal. After each measurement, some of the animals were killed with an intracardiac perfusion of fixative, temporal bones were removed and were immunohistochemically examined for inducible nitric oxide synthase (iNOS/NOS II). On serial paraffin section, iNOS could be detected first after 3 h in the lateral wall, the supporting cells of the organ of Corti and in cells of the spiral ganglion and was observed up to 12 h. After the injection of LPS, the threshold of compound action potential became significantly worse after 12 h in the LPS group. These changes became evident first at higher frequency (8 kHz). These results suggest that iNOS-generated NO is involved in the cochlea dysfunction under inflammatory conditions.