Regeneration of the peripheral nerves in the lumen of implanted vessels

Regeneration of the rat peripheral nerve in the lumen of implanted aorta was examined over 4 months. The nerve invaded the vessel with its entire trunk and went out of the distal vessel to the adjacent muscles. Inflammatory infiltrations were observed only outside the implant. Only insignificant number of inflammatory elements were inside the lumen. These conditions favored good regeneration of the nerve without forming a neuroma at the end of the cut nerve. The regenerating myelinized fibers formed spiral-like or bush-like terminals on muscle fibers. The model of directed regeneration of the peripheral nerves in the vascular lumen holds promise for rapid and accurate reinnervation of tissues and organs.     

Regeneration of the rat sciatic nerve after various experimental lesions (morphologic and morphometric analysis)

Dynamics of structural restoration of the peripheral nerve (n. ischiadicus) have been studied in the noninbred rats in 3 series of experiments: after local freezing, pinching and cutting with a subsequent connection of the nerve ends by means of an implanted arterial vessel. As demonstrate the methods of light and electron microscopy, myelinization of the nervous fibers in the distal part of the nerve begins between the 10th-20th days after the effect. Further, amount of the myelinated nerve fibers (NF) significantly increases, they become essentially thicker. However, even in the later time of the observation (9 months) most of NF remain thinner than in the control nerve; this demonstrates that the reparative processes take a longer time than it was supposed before. The comparative analysis makes it possible to recommend the cryogenic lesion of the nerve as the most perspective model to study processes of the reparative histogenesis. Certain positive signs of sutureless connection of the cut nerve by means of the implanted arterial vessel are noted for clinical substitution of vast diastases of the nerve.     

Dynamics of the recovery of the structure and function of the sciatic nerve and skin receptors in reinnervation of the hindlimb of white rats

The dynamics of the structural and functional repair of nerve trunks and receptor endings in the rat foot skin was studied on the model of sutureless connection of the damaged nerve by and implanted arterial vessel. Neurohistological and neurophysiological methods were used to show that the anatomical repair of the nerve integrity, maturation of nerve fibers and formation of receptor endings preceded their functional restoration. Terms of myelinization of regenerating axons, their diameters and initial stages of the formation of receptors in the foot skin were established. The restoration of the skin receptor function was shown to occur within 11-13 months after operation.     

Implantation of embryonic brain tissue into regenerating peripheral nerve

Dynamics of development of the cerebral cortex tissue anlage in 17-day-old embryos of Wistar rats, implanted into the sciatic nerve of mature rats with the aim to establish new relay and trophic centers in the regenerating nerve have been studied. By means of certain morphological methods (silver nitrate impregnation after Bielschowsky-Gros, Sudan black, hematoxylin-eosin, toluidine blue after Nissl stainings) it has been stated that the implanted nerve cells not only preserve their viability, but also differentiate from neuroblasts up to young and mature neurons during 2 months. Already in 14 days after the operation there are blood vessels in the implants; by the 2d month massive myelinization of axons begins in the implant. A part of the regenerating myelin fibers of the nerve gets into the implant and branches. In similar cases connections between the implanted neurons and the host peripheral nervous fibers are supposed to be established.     

Limb muscle regeneration in peripheral nerve autografts

In a previous study we demonstrated regenerative growth of extraocular muscle within transplanted peripheral nerve autografts. The present study addresses the feasibility of inducing regeneration of limb muscle within autologous peripheral nerve implants in the gluteus medius of beagles. In six anesthetized animals, a 2-cm segment of the left infraorbital sensory nerve was removed from the nose and implanted between the cut ends of several muscle fascicles in the left gluteus medius. After 4 weeks, the nerve grafts were removed and examined by light and electron microscopy. Muscle fibers were seen surrounded by the epineurium of the implanted nerve along its entire length, growing in parallel with the long axis of the nerve. The regenerating fibers were closely associated with the basal lamina of degenerating myelinated and unmyelinated axons. This study suggests that limb muscle, like extraocular muscle, is capable of organized regenerative growth within peripheral nerve autografts.     

Early innervation of skeletal muscle during tail regeneration in urodele amphibians.

The innervation pattern of skeletal muscles was studied in the normal and regenerating tail of Notophthalmus viridescens. Silver staining for nerve endings and histochemical localization of acetylcholinesterase (AChE) were used for light microscopy. In In normal musculature, AChE positive reactions were localized at the ends of the muscle fibers where they are anchored on connective tissue septa by myotendinous junctions. At this level, silver staining shows nerve terminals forming endplates. During regeneration, positive reactions for AChE appear de novo as dense plates localized at the ends of the newly formed myotubes. The mechanisms involved in the localization of AChE on this surface seem to operate before previous local contacts by nerve terminals. From the ultrastructural data and immunohistochemical results with anti-laminin antibody, these observations suggest that regenerating muscle fibers determine a region of post-synaptic specialization in close relation with the organization of myotendinous regions and basement membrane formation. Nerve-muscle contacts appear at these levels at stage IV (15-20 days after amputation) in the stump and in the rostral part of the regenerate (transition zone). These nerve terminals are provided by the disorganized peripheral nervous system of the injured segment. In the regenerate a similar pattern of AChE reaction can be seen in every myotube, differentiating according to a rostro-caudal gradient. Innervation at the ends of the muscle fibers is in spatiotemporal relation with the exists of the ventral roots from the regenerating nerve cord as the regenerate continues to grow in length.     

Persistence of Disorganized Pathways and Tortuous Trajectories of Regenerating Retinal Fibers in the Adult Newt Cynops pyrrhogaster

The anatomical pathways and trajectories of regenerating retinal fibers within the optic tract and tectum of adult newts were examined, 7 months after transection of the optic nerve. In spite of restoration of retinotopic ordered central connection within the tectum, the pathways of individual regenerating retinal fibers within the optic tract were greatly disorganized; the misrouted retinal fibers exhibited tortuous trajectories on the tectal surface in approach to their sites of normal innervation. These results suggest that the regenerating retinal fibers with abnormal pathways and tortuous trajectories can be maintained provided they are in contact with appropriate targets.     

Construction of a physical model of the human carotid artery based upon in vivo magnetic resonance images

A method is described for construction of an in vitro flow model based on in vivo measurements of the lumen geometry of the human carotid bifurcation. A large-scale physical model of the vessel lumen was constructed using fused deposition modeling (a rapid prototyping technique) based on magnetic resonance (MR) images of the carotid bifurcation acquired in a healthy volunteer. The lumen negative was then used to construct a flow model for experimental studies that examined the hemodynamic environment of subject-specific geometry and flow conditions. The physical model also supplements physician insight into the three-dimensional geometry of the arterial segment, complementing the two-dimensional images obtained by MR. Study of the specific geometry and flow conditions in patients with vascular disease may contribute to our understanding of the relationship between their hemodvnamic environment and conditions that lead to the development and progression of arterial disease.     

Restoration of the innervation of the extremity of the rat after joining the ends of the damaged nerve with a microsurgical suture

Morphofunctional restoration of the cut ischemic nerve has been studied after its connection by means of a microsurgical suture. The investigations have been performed on 20 male rats. In 6 months after the operation the average diameter of the regenerating nervous fibers is 5.0-6.5 mcm and, as in an intact nerve they have a wavy course. Myelin decay products and inflammatory infiltrates are revealed in the nerve but sometimes. All the parts of the plantar skin are well reinnervated. Cutaneous epidermis of the sole and the digital pads possesses an extremely rich innervation. Characteristics of M-responses, registered from the m. gastrocnemius, approaches the norm in 2 months after the operation. In 10% of fibers of the regenerating ischiatic nerve impulse activity begins to be registered in 20-30 days after the operation. Gradually the borders of the receptor fields become wider and in 3 months they spread all over the whole sole. In 5-6 months the impulse reaction of the regenerating nerve fibers does not differ from that in non-operated animals. Thus, use of the microsurgical suture results in a successful regeneration of the rat ischemic nerve and in a rather short time. The restorative dynamics of the extremity tissue innervation can be presented as following: at first somatic muscles get innervation and then, as the afferent fibers grow in the periphery, the plantar skin and the skin of the digital pads is the last to get innervation.     

Nerve fiber interrelationships with the ependyma of the 3d ventricle in rats in the perinatal period]

The localization and ultrastructure of nerve fibers in the wall and lumen of ventral region of III ventricle was studied from the 16th day of prenatal period till the 9th day of postnatal period in the Wistar rats. The nerve fibers were first found in the subependyme zone; between the ependyme cells and in the lumen of ventricle on the 18th day of development. The nerve fibers occur constantly in the lumen of ventricle beginning from the 3rd day of postnatal period. The dilatations of nerve fibers contain granules of 50 to 90 nm in diameter with the electron-dense center (carriers of monoamines and/or releasing hormonnes) granules with moderately osmiophilic contents and light vesicles of the same size. It is suggested that these structures reflect different stages of the release of neurohormones in the cerebrospinal fluid. The synapse-like contacts of nerve fibers with the ependyme cells are observed beginning from the 3rd day of postnatal period. Their role in the regulation of absorption of substances by the ependyme cells functioning is discussed.     

Three dimensional observation of the nerve fibers along the cerebral blood vessels

Three dimensional observation of the nerve fibers along the cerebral blood vessels was investigated by scanning electron microscopy. Electron probe X-ray microanalysis was also performed in the cerebral blood vessels treated with peroxidase-antiperoxidase immunohistochemistry intensified by nickel ammonium sulfate. Nerve fibers (2-8 microns in diameter) formed a plexus on the outer surface of the adventitia. After branching, the nerve fibers penetrated the blood vessel adventitia. Substance P-immunoreactive nerve fibers showed a meshwork pattern in the outer layer of the adventitia, and vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers revealed a spiral running pattern in the inner layer of the adventitia. Taken together with previous studies, these findings suggest that substance P nerve fibers in the cerebral arteries may not be related to arterial dilatation or constriction, but VIP nerve fibers may be vasodilative.     

Rate of Movement and Composition of Rapidly Transported Proteins in Regenerating Olfactory Nerve

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 +/- 9 mm/day at 23 degrees, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a light axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high-molecular-weight polypeptides.     

Flow-tissue interaction with compliance mismatch in a model stented artery

The insertion of an endovascular prosthesis is known to have a thrombogenic effect that is also a consequence of the interaction between the flowing blood and the stented arterial segment; in fact the prosthesis induces a compliance mismatch and a possible small expansion along the vessel that eventually gives rise to an anomalous distribution of wall shear stresses. The fluid dynamics inside a rectilinear elastic vessel with compliance and section variation is studied here numerically. A recently introduced perturbative approach is employed to model the interaction between the fluid and the elastic tissue; this approximate technique is first validated by comparison with a complete solution within a simple one-dimensional model of the same system. Then it is applied to an axisymmetric model in order to evaluate the flow dynamics and the distribution of wall shear stress in the stented vessel. Compliance mismatch is shown to produce more intense negative wall shear stresses in the stented segment while rapid variations of wall shear stress are found at the stent ends. These effects are enhanced when the prosthesis is accompanied by a small increase of the vessel lumen.     

Mathematical modeling of the response of a resistive vessel to pressure

The response of small arterial vessels to internal pressure makes an essential contribution to autoregulation in the vascular bed. It is believed that free cytosolic Ca²⁺ concentration plays a pivotal role in the regulation of smooth muscle contractility and hence of the vascular lumen. A simple mathematical model of blood flow in a resistive vessel is suggested. The model is based on the experimental data obtained for cerebral arteries, but may be used for any other resistive vessel. The model not only describes the regulation of the vascular lumen by transmural pressure but also shows realistic behavior of the vessel radius and cytosolic [Ca²⁺] at different rates of pressure change. Possible variations in the radius along the vessel due to the Bayliss effect are considered.     

Quantitative studies on the regeneration of myelinated nerve fibers. II. Variations of the number and size of regenerating nerve fibers after localized crushing or total section]

1. The number and size of myelinated nerve fibers have been determined at standard levels, in the nerve to medial head of right and left gastrocnemius muscles of 112 rats in which the left sciatic nerve had suffered an experimental lesion according one of the following four modalities: localized crushing, total section followed or not by suture and resection of a nervous segment of about 1 cm. 2. In the nerve to medial head of right gastrocnemius muscle (contralateral nerve used as control), the number of myelinated fibers decreased in average to 10% after crushing, 5% or 4% after section followed or not by suture. However, an increase of 6% was observed after resection. The mean values of the mean diameters showed a decrease of 8% after crushing and 5% after section without suture. This value did not seem to be affected by section followed by immediate suture and after resection, it increased of 11%. On the whole, male rats appeared to be more sensitive than female to the effects of the operation. 3. The nerves of 12 rats have been observed from 15 to 334 days after resection of about 1 cm of sciatic nerve. The 20% of the regenerating myelinated nerve fibers which have succeeded to cross over such a distance had a distribution which remained unimodal; the diameter of the large fibres did not exceed 8 micronm. 4. 34 rats have been sacrificed from 15 to 715 days after sciatic nerve section which was not followed by suture. The number of myelinated nerve fibers became normal again during the 4th month and reached afterwards a mean value of 130%, with very marked variations. The nerve fibre distribution was most frequently unimodal, but may came bimodal one year after the operation in certain nerves. Their mean diameter never exceeded 60% of the normal. 5. The nerves of 34 rats have been examined from 15 to 720 days after section and immediate suture. The number of myelinated nerve fibers returned to normal during the second month and increased afterwards to an average of 150% with very important variations. The nerve fiber distribution was generally unimodal, but may become bimodal 7 months after the operation. Their mean diameter reached only 50 to 55% of the normal. 6. 32 rats have been sacrificed from 10 to 720 rats after a localized crushing. The number of myelinated nerve fibers come back to normal during the 4th week and later increased up to a mean of 115%. Their distribution became early bimodal from the 97th day onwards. Although, their mean diameter nerver exceeded 80% of the normal, the histograms of the regenerating nerve and of the control nerve could be almost superposed during the second year.     

Study of regeneration in the garfish olfactory nerve

Previous studies of the olfactory nerve, mainly in higher vertebrates, have indicated that axonal injury causes total degeneration of the mature neurons, followed by replacement of new neuronal cells arising from undifferentiated mucosal cells. A similar regeneration process was confirmed in the garfish olfactory system. Regeneration of the nerve, crushed 1.5 cm from the cell bodies, is found to produce three distinct populations of regenerating fibers. The first traverses the crush site 1 wk postoperative and progresses along the nerve at a rate of 5.8 +/- 0.3 mm/d for the leading fibers of the group. The second group of fibers traverses the crush site after 2 wk postcrush and advances at a rate of 2.1 +/- 0.1 mm/d for the leading fibers. The rate of growth of this group of fibers remains constant for 60 d but subsequently falls to 1.6 +/- 0.2 for the leading population of fibers. The leading fibers in the third group of regenerating axons traverse the crush site after 4 wk and advance at a constant rate of 0.8 +/- 0.2 mm/d. The multiple populations of regenerating fibers with differing rates of growth are discussed in the context of precursor cell maturity at the time of nerve injury and possible conditioning effects of the lesion upon these cells. Electron microscopy indicates that the number of axons decreases extensively after crush. The first two phases of regenerating axons represent a total of between 6 and 10% of the original axonal population and are typically characterized by small fascicles of axons surrounded by Schwann cells and large amounts of collagenous material. The third phase of fibers represents between 50 and 70% of the original axonal population.     

Three dimensional observation of the nerve fibers along the cerebral blood vessels

Summary Three dimensional observation of the nerve fibers along the cerebral blood vessels was investigated by scanning electron microscopy. Electron probe X-ray microanalysis was also performed in the cerebral blood vessels treated with peroxidase-antiperoxidase immunohistochemistry intensified by nickel ammonium sulfate.Nerve fibers (2–8 m in diameter) formed a plexus on the outer surface of the adventitia. After branching, the nerve fibers penetrated the blood vessel adventitia. Substance P-immunoreactive nerve fibers showed a meshwork pattern in the outer layer of the adventitia, and vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers revealed a spiral running pattern in the inner layer of the adventitia. Taken together with previous studies, these findings suggest that substance P nerve fibers in the cerebral arteries may not be related to arterial dilatation or constriction, but VIP nerve fibers may be vasodilative.     

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules.While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.     

Efficiency of reinnervation of neonatal rat muscle of original and foreign nerves

Formation of neuromuscular connections in mammals may involve a hierarchy of efficiency of synapse formation at a stage when motor nerves have already contacted muscle fibers and during the transitional period of multiple innervation. In an attempt to test for such a hierarchy, we examined, in neonatal rats, the relative efficiency of reinnervation by foreign or original nerves implanted simultaneously in a large muscle so that competition for muscle fibers was minimized. The tibial nerve, containing gastrocnemius nerve fibers, and the “foreign” peroneal nerve were implanted into the denervated lateral gastrocnemius muscle. One to five months later, indirect tetanic tensions obtained upon stimulating the implanted nerves were measured by isometric techniques and were compared to contralateral control muscles. When both nerves were implanted side by side at the end-plate region, approximately equal tetanic tensions were obtained at the time of testing. The same result was also obtained when the tibial and common peroneal nerves were implanted into non-end-plate and end-plate regions, respectively. However, in the reverse experiment, the tibial nerve implanted at the end-plate region produced significantly higher tetanic tension than the peroneal nerve at the non-end-plate site in the same muscle. Thus, the original nerve, compared to a foreign nerve, appeared to reinnervate neonatal muscle more effectively, but this was only revealed under conditions where access to former end-plate regions was unequal.     

Progressive reorganization of the myenteric plexus during one year following reanastomosis of the ileum of the guinea pig

The enteric nervous system appears to play a pivotal role in the functional recovery of the gastrointestinal tract after partial resection and reanastomosis, but the structural changes following surgery are not fully understood. The present study was designed to clarify the processes of myenteric plexus regeneration up to one year after transection and reanastomosis of the ileum of the guinea pig. The following techniques were used: nicotinamide adenine dinucleotide (NADH) diaphorase histochemistry, immunostaining of neuron-specific enolase (NSE) in whole-mount preparations, and transmission electron microscopy. Two months after transection and reanastomosis, myenteric ganglion cells with NADH diaphorase reactions were scarce in the center of the lesion, and were less numerous in adjacent areas (3 mm in width) than in the control ileum. In the areas adjacent to the lesion, a few large extraganglionic neurons that did not completely compensate for the loss of ganglion neurons were observed. The remaining ileum showed no changes in NADH diaphorase staining pattern at this stage. Two to 12 months after transection and reanastomosis, ectopic large neurons gradually increased in number not only in the areas adjacent to the lesion but also in part of the remaining ileum, up to 10 cm from the lesion. Concomitantly, large ganglion neurons decreased in number in these areas. In other ileal regions (more than 10 cm distant from the site of transection), no obvious changes in NADH diaphorase staining were noted throughout the observation period. The outgrowth of NSE-containing nerve fibers from the severed stumps was seen two weeks after transection. Six weeks later, numerous bundles of fine nerve fibers with NSE were shown to interconnect the oral and anal cut ends of the myenteric plexus, but they exhibited no subsequent alterations. Transmission electron microscopy revealed that regenerating nerve fiber bundles appeared initially among irregularly arranged smooth muscle cells eight weeks after the operation, as expected from light-microscopic observations. These findings suggest that myenteric ganglion cell bodies, unlike myenteric nerve fibers, require a longer term of reconstruction than previously believed after transection and reanastomosis of the ileum of the guinea pig.