Neurobiological effects of microwave exposure: a review focused on morphological findings in experimental animals.

The possible risk of electromagnetic radiation (EMR) for nervous system is regularly published from the middle of 20th century. Numbers of neurobiological studies demonstrate that various EMR frequencies induce changes in nervous tissue of experimental animals but the evidence for health effect of EMR to the nervous system remains uncertain. To solve the fundamental questions about possible health hazard of modern technologies, the main producers of EMR, further intensive experimental studies on animals are needed. This review, focused on morphological findings achieved in various experimental animals, demonstrates that blood-brain barrier is the most studied morpho-functional unit of CNS in experiments with EMR. The morphological findings in experimental animals, in many cases controversial, put some evidence on nervous tissue structural damage after the EMR exposure. In spite of numerous literary data a wide range of contemporary neuro-morphological methods waits to be utilized in the EMR experimental paradigm. Using these methods could play an important role in answering the question about possible adverse effects of microwaves on nervous system.

Fluoro-Jade B evidence of induced ischemic tolerance in the rat spinal cord ischemia: physiological, neurological and histopathological consequences.

Fluoro-Jade B, a marker of degenerating neurons, was used to label histopathological changes in the rat spinal cord after transient ischemia and ischemic preconditioning (IPC). To characterize postischemic neurodegenerations and consequent neurological changes, a particular attention was paid to the standardization of ischemic conditions in animals of both groups. 1. The control ischemic rats were submitted to a reversible occlusion of descending aorta by insertion and subsequent inflation of a 2F Fogarty catheter for 12 min. 2. In the IPC rats, an episode of short 3 min occlusion and 30 min reperfusion preceded the 12 min ischemia. Postischemic motor function testing (ambulation and stepping) was provided repeatedly for evaluation of neurological status 2 h and 24 h after surgery and at the end of postischemic survival, i.e. after 48 h. Fluoro-Jade B staining was used to demonstrate degenerated neurons. In the control rats, neurological consequences of histopathological changes in lumbosacral spinal cord, manifested as paraplegia, were present after 12 min ischemia. Thus, numbers of degenerated Fluoro-Jade B positive cells were visible in gray matter of the most injured L(4)-S(2) spinal cord segments. Slight motor function impairment, consequential from significant decreasing in Fluoro-Jade B-positivity in the L(4)-S(2) spinal cord segments of the IPC rats, was considered the pathomorpfological evidence that IPC induces spinal cord tolerance to ischemia. Our results are consistent with the previously published silver impregnation method for histopathological demonstration of ischemic degeneration.

Cauda equina syndrome.

Single or double-level compression of the lumbosacral nerve roots located in the dural sac results in a polyradicular symptomatology clinically diagnosed as cauda equina syndrome. The cauda equina nerve roots provide the sensory and motor innervation of most of the lower extremities, the pelvic floor and the sphincters. Therefore, in a fully developed cauda equina syndrome, multiple signs of sensory disorders may appear. These disorders include low-back pain, saddle anesthesia, bilateral sciatica, then motor weakness of the lower extremities or chronic paraplegia and, bladder dysfunction. Multiple etiologies can cause the cauda equina syndrome. Among them, non-neoplastic compressive etiologies such as herniated lumbosacral discs and spinal stenosis and spinal neoplasms play a significant role in the development of the cauda equina syndrome. Non-compressive etiologies of the cauda equina syndrome include ischemic insults, inflammatory conditions, spinal arachnoiditis and other infectious etiologies. The use of canine, porcine and rat models mimicking the cauda equina syndrome enabled discovery of the effects of the compression on nerve root neural and vascular anatomy, the impairment of impulse propagation and the changes of the neurotransmitters in the spinal cord after compression of cauda equina. The involvement of intrinsic spinal cord neurons in the compression-induced cauda equina syndrome includes anterograde, retrograde and transneuronal degeneration in the lumbosacral segments. Prominent changes of NADPH diaphorase exhibiting, Fos-like immunoreactive and heat shock protein HSP72 were detected in the lumbosacral segments in a short-and long-lasting compression of the cauda equina in the dog. Developments in the diagnosis and treatment of patients with back pain, sciatica and with a herniated lumbar disc are mentioned, including many treatment options available.

Fos protein expression in sacral spinal cord in relation to early phase of cauda equina syndrome in dogs.

1. The aim of the present study is to map the incipient phase of Fos expression in the sacral spinal cord neuronal pools of multiple cauda equina constrictions canine model. 2. Fos-positive neurons were found bilaterally in the lateral portion of superficial dorsal horn layers (Laminae I-III) and along the lateral edge of the dorsal horn accompanied by the lateral collateral pathway, fibers of Lissauer's tract, terminating at the sacral parasympathetic nucleus. Similarly, high Fos expression was detected in the ventral portion of the dorsal sacral commissure and in the dorsomedial portion of the anterior horns at S1-S3 segment level. Finally, a clearly expressed Fos-positivity was disclosed bilaterally in the neuropil of the nucleus Y in the anterior horn. 3. Data from the present study show that continuous stimulation of the central fibers of sacral dorsal root ganglia neurons, i.e., fibers of sacral primary afferents, unlike those using various stimulations of the peripheral fibres offers an unusual pattern of Fos-like immunoreactivity.

Transient spinal cord ischemia in rat: the time course of spinal FOS protein expression and the effect of intraischemic hypothermia (27 degrees C).

1. In the present study, we characterize the time course of spinal FOS protein expression after transient noninjurious (6-min) or injurious (12-min) spinal ischemia induced by inflation of a balloon catheter placed into the descending thoracic aorta. In addition, this work examined the effects of spinal hypothermia on FOS expression induced either by ischemia or by potassium-evoked depolarization (intrathecal KCl). 2. Short-lasting (6-min) spinal ischemia evoked a transient FOS protein expression. The peak expression was seen 2 hr after reperfusion in all laminar levels in lumbosacral segments. At 4 hr of reperfusion, more selective FOS expression in spinal interneurons localized in the central part of laminae V-VII was seen. At 24 hr no significant increase in FOS protein was detected. 3. After 12 min of ischemia and 2 hr of reflow, nonspecific FOS expression was seen in both white and gray matter, predominantly in nonneuronal elements. Intrathecal KCl-induced FOS expression in spinal neurons in the dorsal horn and in the intermediate zone. Spinal hypothermia (27 degrees C) significantly suppressed FOS expression after 6 or 12 min of ischemia but not after KCl-evoked depolarization. 4. Data from the present study show that an injurious (but not noninjurious) interval of spinal ischemia evokes spinal FOS protein expression in glial cells 2 hr after reflow. The lack of neuronal FOS expression corresponds with extensive neuronal degeneration seen in this region 24 hr after reflow. Noninjurious (6-min) ischemia induced a transient, but typically neuronal FOS expression. The significant blocking effect of hypothermia (27 degrees C) on the FOS induction after ischemia but not after potassium-evoked depolarization also suggests that simple neuronal depolarization is a key trigger in FOS induction.

Incipient cauda equina syndrome as a model of somatovisceral pain in dogs: spinal cord structures involved as revealed by the expression of c-fos and NADPH diaphorase activity.

Segmental and laminar distribution of Fos-like immunoreactive, reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting and double-labeled (Fos-like immunoreactive and NADPHd-exhibiting) neurons was examined in lower lumbar and sacral segments of the dog spinal cord using the model of multiple cauda equina constrictions. NADPHd histochemistry was used as marker of nitric oxide synthase-containing neurons. The appearance and the time-course of Fos-like immunoreactive, NADPHd and double-labeled neurons was studied at 2 h and 8 h postconstriction characterized as the incipient phase of cauda equina syndrome. The occurrence of Fos-like immunoreactive and NADPHd-exhibiting neurons in fully developed cauda equina syndrome was studied at five days postconstriction. An increase in Fos-like immunoreactivity in superficial laminae (I-II) and an enhanced NADPHd staining of lamina VIII neurons were found. A statistically significant increase in Fos-like immunoreactive neurons was found in laminae I-II and VIII-X 8 h postconstriction, and in contrast, a prominent decrease in Fos-like immunoreactive neurons was found in laminae I-II, accompanied by a statistically significant increase in Fos-like immunoreactive neurons in more ventrally located laminae VII-X at five days postconstriction. Quantitative analysis of laminar distribution of constriction-induced NADPHd-exhibiting neurons revealed a considerable increase in these neurons in laminae VIII-IX 8 h postconstriction and a statistically highly significant increase in NADPHd-exhibiting neurons in laminae VII-X five days postconstriction. Concurrently, the number of NADPHd-exhibiting neurons in laminae I-II was greatly reduced. While a low number of double-labeled neurons was found throughout the gray matter of lower lumbar and sacral segments at 2 h postconstriction, a statistically significant number of double-labeled neurons was found in lamina X 8 h and in laminae VII-X five days postconstriction. The course and distribution of anterograde degeneration resulting five days after multiple cauda equina constrictions are compared with segmental and laminar distribution of Fos-like immunoreactive and NADPHd-exhibiting neurons. Prominent involvement of the spinal cord neurons appearing in the lumbosacral segments at the early beginning and in fully developed cauda equina syndrome results in a Fos-like immunoreactivity and strongly enhanced NADPHd staining of some neuronal pools. Under such circumstances, an early cauda equina decompression surgery is advisable aimed at decreasing or preventing the derangement of the neural circuits in the lumbosacral segments.

[Neural stem cells: possibilities of regeneration in the adult CNS]. / Neurálne kmenové bunky: perspektívy regenerácie v dospelom CNS.

The persistence of neurogenesis and structural plasticity of the nervous tissue was believed until recently to be restricted to lower vertebrates and songbirds. Nevertheless, it has now been ascertained that these phenomena can occur in the adult mammalian nervous system, at least in three distinct sites: the olfactory neuroepithelium of the nasal mucosa and two brain regions, namely, the hippocampal dentate gyrus and the olfactory bulb. The presented review addresses the nature, distribution and regulation of neural stem cells and the prospect for using these cells in therapeutic strategies of the damaged CNS reparation. (Fig. 2, Ref. 30.)

Reduced nicotinamide adenine dinucleotide phosphate diaphorase in the spinal cord of dogs.

The distribution of somatic, fibre-like and punctate, non-somatic reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity was examined in dog spinal cord using horizontal, sagittal and transverse sections. The morphological features of NADPH diaphorase exhibiting neurons divided into six different neuronal types (N1-N6) were described and their laminar distribution specified. Major cell groups were identified in the superficial dorsal horn and around the central canal at all spinal levels, and in the intermediolateral cell column at thoracic level. NADPH diaphorase exhibiting neurons of the pericentral region were distributed in a thin subependymal cell column containing longitudinally-arranged small bipolar neurons with processes penetrating deeply into the intermediolateral cell column and/or running rostrocaudally in the subependymal layer. The second pericentral cell column located more laterally in lamina X contains large, intensely-stained NADPH diaphorase exhibiting neurons with long dendrites radiating in the transverse plane. Neurons of the sacral parasympathetic nucleus seen in segments S1-S3 exhibited prominent NADPH diaphorase activity accompanied by heavily-stained fibres extending from Lissauer's tract through lamina I along the lateral edge of the dorsal horn to lamina V. A massive dorsal gray commissure, with high NADPH diaphorase activity, was found in segments S1-S3. At the same segmental level a prominent group of moderately-stained motoneurons was detected in the dorsolateral portion of the anterior horn. Fibre-like NADPH diaphorase activity was found in the superficial dorsal horn and pericentral region in all segments studied. Punctate, non-somatic NADPH diaphorase activity was detected in the superficial dorsal horn, in the pericentral region all along the rostrocaudal axis and in the nucleus phrenicus (segments C4-C5), nucleus dorsalis (segments Th2-L2), nucleus Y (segments S1-S3), and the dorsal part of the dorsal gray commissure (S1-S3). A schematic diagram documenting the segmental and laminar distribution of NADPH diaphorase activity is given.

Knee joint inflammation attenuates spinal FOS expression after unilateral paw formalin injection in rat.

Carrageenan-induced knee joint inflammation evoked a transient spinal FOS protein expression in neurons localized in the apical region of laminae I-III with peak activity observed 2 h after inflammation. Consistent with previously published observations, paw formalin injection evoked a distinct pattern of FOS protein expression in L3-L5 spinal segments. The majority of FOS-positive neurons were localized in the superficial dorsal horn (laminae I-II). Laminae V and VI contained moderate numbers of labeled neurons and only a few labeled nuclei were visible in laminae VII-X. In contrast, ipsilateral paw formalin injection, if administered 4 h after carrageenan-induced knee inflammation, evoked significantly fewer FOS positive neurons in all laminar and segmental levels analyzed as compared with formalin injected animals but without previous knee joint inflammation. These data indicate that primary acute or subacute nociceptive input may evoke central processes that are characterized by an inducible form of central inhibition which then may serve to modulate the subsequent spinal effect of superimposed nociceptive peripheral stimulation.

Multiple protracted cauda equina constrictions cause deep derangement in the lumbosacral spinal cord circuitry in the dog.

Neuropathological changes of the neuronal pools and spinal cord circuitry in the lumbosacral segments were studied in a canine model of multiple protracted cauda equina constrictions. Anterograde degeneration of all sacrococcygeal and L7 dorsal root fibers was detected in S1-S3 and lower lumbar segments. A narrow degenerated gracile fascicle was found in all thoracic and cervical segments terminating in the gracile nucleus. Transneuronal degeneration of middle-sized and large neurons, located in S1-S3 and sporadically in L7 segments, was noted. Identical transneuronal degeneration was seen in a group of small neurons located in the ventralmost part of lamina VII in S1-S3 segments. Simultaneously, a terminal degeneration was detected in the lateral cervical nucleus and in the ventral posterior lateral nucleus of the ventrobasal thalamic complex. Concomitantly, a fully developed retrograde degeneration affecting motoneurons in the ventrolateral portion of the anterior horn in S1-S3 segments appeared.

Formation of filamentous and multivesicular bodies after intrathecal MK-801 injection.

Our study of ultrastructural changes in lumbar spinal cord is based on the rats permanent intrathecal (IT) catheter model of MK-801 injection. After 3 days survival with the IT catheter the animals were treated by injection of MK-801 or saline. Five hrs post-treatment the rats were transcardially perfused with 4% paraformaldehyde in 0.1 M PB. L4-5 segments of the spinal cord were dissected and processed for EM. The presence of single or multiple filamentous/multivesicular bodies in neuronal cytoplasm of MK-801 treated animals was apparent.

Ischemia-induced delayed-onset paraplegia is accompanied by an unusual form of synaptic degeneration in the lumbosacral segments: an experimental light and electron microscopic study in dogs.

We studied the effect of high thoracic aorta cross-clamping, complete transverse section of the spinal cord at Th6 level, and combined hemisection at Th6 level followed later by high thoracic aorta cross-clamping upon the morphology and number of identified presynaptic knobs in lumbosacral segments in dogs. In animals surviving 48-72 hours after high thoracic aorta cross-clamping the occurrence of an unusual form of boutons accompanied by periboutonal halo in L3-S1 segments was found. According to the bouton size and light as well as electron microscopic appearance, four types, i.e., light giant (T1), dark enlarged (T2), light giant with periboutonal halo (T3), and giant disintegrating (T4) boutons were detected after 48 and 72 hour reperfusion. The appearance of four boutonal types in the lumbosacral segments is caused by spinal cord ischemia secondary to high thoracic aorta cross-clamping followed by 48 or 72 hour reperfusion. At the end of the sixth reperfusion day no signs of enlarged and giant boutons were detected in L3-S1 segments. A statistically significant increase of enlarged and giant boutons was noted at the end of the third reperfusion day in comparison with 48 hour survival. After spinal cord transection at midthoracic (Th6) level, followed by 72 hour survival, no such unusual synaptic knobs could be found in L3-S1 segments. The laminar distribution pattern of T1-T4 types based on light microscopic analysis and confirmed electron microscopically is characteristic and strictly bound to those spinal cord gray matter layers which serve as main termination sites of the descending cortical, brain stem, as well as long propriospinal projections in the lumbosacral segments (laminae V-VII). A statistically significant increase of enlarged and giant boutons was found in the intermediate zone (lamina VII). Hemisection at midthoracic level (Th6) followed later by 30 minute high thoracic aorta cross-clamping and 48 hour reperfusion caused a marked decrease of enlarged and giant boutons in L3-S1 segments on the hemisectioned side in comparison with the contralateral one. Large amounts of irregularly arranged round vesicles and tubular profiles were disclosed in the boutonal matrix of T1, T3, and T4 types in L3-S1 segments of animals subjected to 30 minute high thoracic aorta cross-clamping followed by 72 hour reperfusion. Accumulation of tubular and membranous materials was invariably seen in the bulbous enlargement of the terminal axonal branch.(ABSTRACT TRUNCATED AT 400 WORDS)

A new applicability of the suppressive Nauta method in the early phase of neuronal damage.

A new applicability of the suppressive Nauta impregnation method was tested allowing the detailed mapping of early neuronal damage expressed in the form of somatodendritic argyrophilia. Two spinal cord ischemia-reperfusion models of rabbit and dog, a model of canine global brain ischemia-reperfusion, involving cardiac arrest followed by resuscitation, and a canine spinal cord compression-decompression model were used. Early phases of neuronal damage are characterized by conspicuous somatodendritic argyrophilia permitting an exact evaluation of acute neuronal damage according to soma size, dendritic ramifications and localization of the affected neuronal neuronal pool. By its high sensitivity to somatodendritic damage the suppressive Nauta method appears to be a valuable neuropathological technique.

Transplantation of dorsal root ganglion into the olfactory bulb of neonatal rats: a histochemical study.

We have transplanted encapsulated dorsal root ganglia (DRG) from adult Wistar albino rats unilaterally into partially bulbectomized (n = 20) neonatal (P3-5) rats of the same strain. Three months postoperatively the animals were perfused and their brains processed by direct thiocholine method for cholinesterases (Ch), specific acetylcholinesterase (AChE) and nonspecific butyrylcholinesterase (BuChE) or stained by Cresyl violet. Selected sections were immunohistochemically stained for olfactory marker protein (OMP). In 17 cases we found surviving transplanted DRG. Fifteen transplants were well integrated with the spared portion of the olfactory bulb (OB) as clearly demonstrated by AChE and BuChE histochemistry, while two did not integrate. Regenerated OMP positive olfactory axons originating from neuroepithelium and AChE positive fibres from OB remnant penetrated into the transplants. In one case, fibers connected with BuChE positive Schwann cells grew from the transplanted DRG into the host OB. Individual sensory neurons of the transplants revealed variable intensity of the AChE staining, thus resembling the pattern of AChE activity in normal DRG. BuChE activity was mostly localized on the surface of sensory neurons in the ring of satellite cells. Some BuChE positive blood vessels penetrated into the DRG, and were observed around sensory neurons. The results showed a considerable viability and adaptability of the sensory neurons in the new environment after a long-term transplantation.

Post cardiac arrest hyperoxic resuscitation enhances neuronal vulnerability of the respiratory rhythm generator and some brainstem and spinal cord neuronal pools in the dog.

Selective neuronal vulnerability of the motor cortex, basal ganglia, brainstem, medulla, cerebellum, C6 and L6 segments of the spinal cord were studied after 15 min of cardiac arrest followed by 1 h of normoxic or hyperoxic resuscitation using the suppressive Nauta method in dogs. Hyperoxic resuscitation causes characteristic somatodendritic argyrophilia of the interneuronal pool in the spinal cord and lower medulla. Cuneate, lateral reticular, supraspinal, and caudal trigeminal nuclei as well as the dorsal and ventral respiratory neuronal groups were heavily involved. Similarly, the Purkinje cells, neurons in the middle and deep portions of the mesencephalic tectum, perirubral, pretectal, posterior commissure, middle-sized striatal and giant pyramidal (Betz's) neurons in the motor cortex became argyrophilic. Hyperoxic resuscitation versus normoxic resuscitation causes statistically significant somatodendritic argyrophilia of the dorsal respiratory group, cuneate, dorsal lateral geniculate and thalamic reticular nuclei.

Silver impregnability of ischemia-sensitive neocortical neurons after 15 minutes of cardiac arrest in the dog.

The development of postischemic neuronal argyrophilia and the subsequent fate of argyrophilic neurons were studied in dogs after 15 min of complete cerebral ischemia and survival varying from 1 h to 7 days. Histopathological examination of the vulnerable neocortical region was performed using the Nauta degeneration method, and the time course of cellular changes was described. Clear-cut neuronal argyrophilia was found to precede cell body shrinkage and gradual disintegration corresponding to selective neuronal death. To clarify this initial stage of neuronal impregnability, the samples from the animals surviving 8 h postarrest were stained with toluidine blue or processed for electron microscopy, and the distribution of argyrophilic cells was confirmed to be identical with that of hyperchromatic or electron-dense cells. On the other hand, infrequently observed "tissue infarctions" exhibited no silver affinity in spite of apparent cellular damage. These findings indicate that enhanced impregnability is related to cytochemical processes incidental to the phenomenon of "selective neuronal death", which can be readily detected by the Nauta method.

[Neurotransplantation, critical analysis and perspectives]. / Neurotransplantácie, kritická analýza a perspektívy.

Basic morphological and functional properties of basal ganglia are described and their role in the development of Parkinson's disease is discussed in detail. Based on recent experimental data, the problem of human autologous adrenal medulla transplants as well as human dopaminergic neurons is analyzed and prospective approaches to successful therapeutic interventions are considered mainly from the molecular and immunobiological point of view. The relative limitations of the regenerative capacity and transplantability of the nervous tissue are assessed and compared with the critical developmental periods of human neurogenesis (Fig. 6, Ref. 47.).

The blood-brain barrier permeability in graded postischemic spinal cord reoxygenation in rabbits.

Postischemic blood-brain barrier permeability changes were studied using a rabbit spinal cord ischemia model followed by normoxic recirculation (group I) or graded postischemic reoxygenation (group II). No signs of Evans blue leakage were found in lumbar segments 3 h after normoxic blood recirculation. After 6 h, the fluorescence was apparent in the perivascular space and in the pericytes, followed by a massive penetration of the tracer into the neuropil and perikarya at 12 h survival; 18 h after normoxic reperfusion, the fluorescence was localized in the cytoplasm of the middle-sized and large neurons. Graded postischemic reoxygenation of lumbar segments applied during the same survival periods had a highly protective effect on vascular membrane permeability. The structural components of the vascular wall as well as neuropil and perikarya remained after its application entirely tracer free.

The relation between free and membrane-bound ribosomes in light dorsal root ganglia neurons after aortic ligature in dogs.

To study the relation between free and membrane-bound ribosomes after repeated aortic ligature we examined light dorsal root ganglia (DRG) neurons by quantitative electron microscopy. For counting ribosomes we applied two methods--the hit method and the total counting of ribosomes on electronograms. By both methods we found that there was a prevalence of free ribosomes in DRG neurons on the first and second postoperative day this relation was significantly shifted in favour of free ribosomes which was in agreement with our morphological findings characterized by disaggregation of polysomes and an increasing number of membrane-free ribosomes. At the advanced period of survival (6 days) the studied relation no longer differed from control values. These findings indicate that reorganized proteosynthesis in light DRG neurons plays a most important role during recovery after ischemic insult.