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.     

Immunocytochemical study of the hepatic innervation in the rat after partial hepatectomy

Summary The autonomic nervous system in rats has been assessed by means of indirect immunofluorescence using monospecific antibodies to neuron-specific enolase, neurofilaments, glial fibrillary acidic protein and S-100 protein (10 days after partial (70%) hepatectomy). Different groups of rats were studied:group A: 70% resection and normal dual blood supply (n=5);group B: 70% resection with only portal blood to the liver remnant (n=5);group C: 70% resection with only arterial blood to the liver remnant (n=5);group D: sham operated controls (n=5).All rats of groups A and D showed normal liver/body weight ratios after 10 days in contrast to groups B and C where liver weights were 50–60% of the preresection weight. In group A the regeneration process was histologically normal and associated with a remarkable increase of autonomic innervation patterns in the portal triad. In contrast, livers of animals in groups B and C showed under the light microscope features of hepatocyte degeneration associated with a decreased autonomic innervation compared to the controls. The changes are identical in groups B and C, and are therefore irrespective of the type of blood deprivation (arterial or portal).These results support the importance of dual blood supply for an optimal regenerative response in liver remnants after liver resection. We suggest that the autonomic nerve supply of the portal triad plays at least an important permissive role in liver regeneration.     

Degenerative and regenerative processes in the olfactory system of homing pigeons.

Experimental resection of the olfactory nerve in the homing pigeon induces a total degeneration of the nerve and olfactory epithelium. The orthograde degenerative process starts before the retrograde one. Ten days after resection, new neurons begin to differentiate from the basal cells. The axon forms earlier than the distal dendritic process, and the speed of growth increases slowly. The regenerated axons only reach the bulb in the 5th month. Two months after resection the olfactory epithelium is similar to that of the intact control side. The ultrastructural features of the mucosa and olfactory axons are similar to those of normal ones.     

Functional status of the regenerated chorda tympani nerve as assessed in a salt taste discrimination task

We tested whether the recovered ability of rats to discriminate NaCl from KCl after chorda tympani nerve transection (CTX) is causally linked to nerve regeneration or some other compensatory process. Rats were presurgically trained in an operant NaCl vs. KCl discrimination task. Rats with regenerated nerves, histologically confirmed by anterior tongue taste pore counts and tested 62 days after CTX (CTX-62R; n = 5), performed as well as those tested 62 days after sham surgery (Sham-62; n = 5), but both of these groups initially performed slightly worse than animals tested 7 days after sham surgery (Sham-7; n = 4). Performance of rats tested either 7 (CTX-7P; n = 5) or 62 (CTX-62P; n = 4) days after CTX in which nerve regeneration was prevented was severely disrupted. Adulteration of the stimuli with amiloride, an epithelial sodium channel blocker, impaired discrimination performance in a similar dose-dependent manner in the Sham-7 (n = 2), Sham-62 (n = 5), and CTX-62R (n = 5) groups, suggesting that the functional status of the amiloride-sensitive transduction pathway returns to normal in rats with regenerated chorda tympani nerves. Performance of CTX rats without regenerated nerves (CTX-7P, n = 2; CTX-62P, n = 4) was further degraded by amiloride treatment, suggesting that taste receptors innervated by other nerves are sensitive to amiloride. In conclusion, nerve regeneration is an essential component underlying full recovery of salt discrimination function after CTX.     

Nerve growth factor expression response to induction agent booster dosing in transfected human embryonic kidney cells

To assess whether nerve growth factor (NGF) expression would respond to booster dosing with the inducing agent ponasterone A, human embryonic kidney cells (HEK-293) were transfected with human NGF cDNA. Cells were cultured for 5 days in media with or without ponasterone A. On day 5, controls received a ponasterone A media replacement, whereas experimental groups received ponasterone A booster media replacement. NGF protein expression bioactivity was assessed using a PC-12 cell bioassay and the concentration of secreted NGF was quantified using NGF enzyme-linked immunosorbent assay. Cells with and without ponasterone A were left for 5 days without changing the medium. On day 5, the supernatants were collected and flash-frozen for enzyme-linked immunosorbent assay. The ponasterone A-positive and -negative booster medium was replaced in the appropriate wells. Supernatants were collected from the wells at 2, 4, and 6 days after the booster dose and removal of original supernatant. The medium was flash-frozen for enzyme-linked immunosorbent assay (1.5 ml), and the remaining 500 mul was transferred to PC-12 cells seeded onto 12-well plates to determine NGF bioactivity. All experiments were performed in quadruplicate. NGF production was measured daily by enzyme-linked immunosorbent assay over a 6-day period after the ponasterone A booster to a maximal release of 1233 +/- 130 pg/ml at day 6 (11 days after original induction). Maximal NGF production per 10(3) cells was 2.5 +/- 0.61 pg at day 6. Bioactivity was determined by percentage differentiation (per 100 cells counted) at 26, 52, and 98 percent for ponasterone A-treated wells on 2, 4, and 6 days after booster dosing (7, 9, and 11 days after induction), respectively. PC-12 cell differentiation was not visualized in the ponasterone A-negative control wells. Human NGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent ponasterone A. Furthermore, repeated bioactive NGF expression peaks beyond that previously demonstrated can be achieved using induction agent booster dosing, indicating the ability to regulate the system over an extended period.     

Enzyme histochemistry of the spinal cord after experimental transection (Th 9) in the cat

Subpial complete resection of a 10 mm segment of the spinal cord at Th 9 was performed in 9 adult cats. Topographic enzyme histochemical investigations of the terminal clubs were performed after different survival times after transection in 7 cats and three days after a subsequent one-week-delayed autologous sciatic grafting procedure in the remaining two cats. For acid phosphatase (ACP), the count of active terminal clubs was high (200 per m2) from 12 hours until day 3 after transection. Then the count of active terminal clubs decreased to a low level (20 per m2) and remained the same until day 14. Removal of necrotic tissue and subsequent grafting with autologous sciatic nerve did not change these findings. For succinate dehydrogenase (SDH), the numbers of terminal clubs showed the same pattern at a lower level. The SDH defined terminal clubs were smaller than the ACP ones. The length of the SDH positive area decreased after 7 days while the ACP positive area remained the same until day 14. The SDH active terminal clubs are overgrown by the ACP positive terminal clubs, after the 7th day. Considering that SDH is linked to constructive activity in mitochondria and ACP to destructive activity in lysosomes, this phenomenon might be responsible for the termination of the capacity of the spinal cord tissue to regenerate.     

Antihyperalgesic effect of simultaneously released hydromorphone and bupivacaine from polymer fibers in the rat chronic constriction injury model

We aimed to evaluate the antihyperalgesic efficacy of a combination of hydromorphone (HM) and bupivacaine (BP) delivered via controlled release from a biodegradable cylindrical rod. In vivo studies were performed using a rat model of thermal hyperalgesia induced by chronic constriction injury (CCI) of the sciatic nerve with loose ligatures. Poly(lactic-co-glycolic acid) (PLGA) rods (10 mm length, 1 mm diameter) loaded with HM (5 mg per rod), BP (5 mg per rod) or no drug (placebo) were implanted subcutaneously, in single or dual pairs, adjacent to the constriction injury, immediately after nerve ligation. We evaluated the efficacy of two dose levels for each drug, alone or in combination, in attenuating thermal hyperesthesia over a period of 12 days according to a prevention protocol. Plasma levels of drugs released from the rods and also released in an in vitro simulation were evaluated. In vitro studies demonstrated that drug release is maintained for at least 10 days. HM (5 mg) alone and BP (5 mg) alone did not attenuate hyperalgesia. Their combination provided a significant increase in the paw withdrawal latency as compared to single agents or placebo. When the dose in each group was doubled, implanting four rods, significant attenuation of hyperalgesia was observed. Analyses of rods retrieved after termination of experiments (after 12 days) revealed 30% residual HM and 70% residual BP content. Prolonged delivery of HM and BP alone or in combination via locally applied PLGA rods may offer a feasible alternative to provide long-lasting analgesia.     

Temporal expression profiles indicate a primary function for microRNA during the peak of DNA replication after rat partial hepatectomy

The liver has the unique capacity to regenerate after surgical resection. However, the regulation of liver regeneration is not completely understood. Recent reports indicate an essential role for small noncoding microRNAs (miRNAs) in the regulation of hepatic development, carcinogenesis, and early regeneration. We hypothesized that miRNAs are critically involved in all phases of liver regeneration after partial hepatectomy. We performed miRNA microarray analyses after 70% partial hepatectomy in rats under isoflurane anesthesia at different time points (0 h to 5 days) and after sham laparotomy. Putative targets of differentially expressed miRNAs were determined using a bioinformatic approach. Two-dimensional (2D)-PAGE proteomic analyses and protein identification were performed on specimens at 0 and 24 h after resection. The temporal dynamics of liver regeneration were characterized by 5-bromo- 2-deoxyuridine, proliferating cell nuclear antigen, IL-6, and hepatocyte growth factor. We demonstrate that miRNA expression patterns changed during liver regeneration and that these changes were most evident during the peak of DNA replication at 24 h after resection. Expression of 13 miRNAs was significantly reduced 12-48 h after resection (>25% change), out of which downreguation was confirmed in isolated hepatocytes for 6 miRNAs at 24 h, whereas three miRNAs were significantly upregulated. Proteomic analysis revealed 65 upregulated proteins; among them, 23 represent putative targets of the differentially expressed miRNAs. We provide a temporal miRNA expression and proteomic dataset of the regenerating rat liver, which indicates a primary function for miRNA during the peak of DNA replication. These data will assist further functional studies on the role of miRNAs during liver regeneration.     

Na,K-ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.     

Median nerve neuropathy in the forearm due to recurrence of anterior wrist ganglion that originates from the scaphotrapezial joint: Case Report

Background

Median nerve neuropathy caused by compression from a tumor in the forearm is rare. Cases with anterior wrist ganglion have high recurrence rates despite surgical treatment. Here, we report the recurrence of an anterior wrist ganglion that originated from the Scaphotrapezial joint due to incomplete resection and that caused median nerve neuropathy in the distal forearm.

Case presentation

A 47-year-old right-handed housewife noted the appearance of soft swelling on the volar aspect of her left distal forearm, and local resection surgery was performed twice at another hospital. One year after the last surgery, the swelling reappeared and was associated with numbness and pain in the radial volar aspect of the hand. Magnetic resonance imaging revealed that the multicystic lesion originated from the Scaphotrapezial joint and had expanded beyond the wrist. Exploration of the left median nerve showed that it was compressed by a large ovoid cystic lesion at the distal forearm near the proximal end of the carpal tunnel. We resected the cystic lesion to the Scaphotrapezial joint. Her symptoms disappeared 1 week after surgery, and complications or recurrent symptoms were absent 13 months after surgery.

Conclusions

A typical median nerve compression was caused by incomplete resection of an anterior wrist ganglion, which may have induced widening of the cyst. Cases with anterior wrist ganglion have high recurrence rates and require extra attention in their treatment.     

Sural Nerve Interposition Grafting during Radical Prostatectomy

In 1997, autologous sural nerve grafting to reconstruct bilaterally resected cavernosal nerves was successfully performed in patients undergoing radical retropubic prostatectomy. After 12 months, one third of these patients had erections sufficient for intercourse. Since that time, patients who have had neurovascular bundle resection and sural nerve grafting have continued to show promising results. For example, within one large cohort of men who had unilateral, nerve-sparing radical prostatectomy, significantly more men who had sural nerve grafting regained potency, and did so in less time, than men who did not have grafting. More importantly, however, with better predictions of the presence of extracapsular disease, nerve-sparing surgery can be performed more selectively, reserving wide resection and sural nerve grafting for patients likely to have extracapsular extension. A multicenter, randomized clinical trial is needed to substantiate the positive outcomes observed with sural nerve grafting.     

Neurovascular free-muscle transfer for the treatment of established facial paralysis following ablative surgery in the parotid region

Neurovascular free-muscle transfer for facial reanimation was performed as a secondary reconstructive procedure for 45 patients with facial paralysis resulting from ablative surgery in the parotid region. This intervention differs from neurovascular free-muscle transfer for treatment of established facial paralysis resulting from conditions such as congenital dysfunction, unresolved Bell palsy, Hunt syndrome, or intracranial morbidity, with difficulties including selection of recipient vessels and nerves, and requirements for soft-tissue augmentation. This article describes the authors' operative procedure for neurovascular free-muscle transfer after ablative surgery in the parotid region. Gracilis muscle (n = 24) or latissimus dorsi muscle (n = 21) was used for transfer. With gracilis transfer, recipient vessels comprised the superficial temporal vessels in 12 patients and the facial vessels in 12. For latissimus dorsi transfer, recipient vessels comprised the facial vessels in 16 patients and the superior thyroid artery and superior thyroid or internal jugular vein in four. Facial vessels on the contralateral side were used with interpositional graft of radial vessels in the remaining patient with latissimus dorsi transfer. Cross-face nerve grafting was performed before muscle transfer in 22 patients undergoing gracilis transfer. In the remaining two gracilis patients, the ipsilateral facial nerve stump was used as the primary recipient nerve. Dermal fat flap overlying the gracilis muscle was used for cheek augmentation in one patient. In the other 23 patients, only the gracilis muscle was used. With latissimus dorsi transfer, the ipsilateral facial nerve stump was used as the recipient nerve in three patients, and a cross-face nerve graft was selected as the recipient nerve in six. The contralateral facial nerve was selected as the recipient nerve in 12 patients, and a thoracodorsal nerve from the latissimus dorsi muscle segment was crossed through the upper lip to the primary recipient branches. A soft-tissue flap was transferred simultaneously with the latissimus muscle segment in three patients. Contraction of grafted muscle was not observed in two patients with gracilis transfer and in three patients with latissimus dorsi transfer. In one patient with gracilis transfer and one patient with latissimus dorsi transfer, acquired muscle contraction was excessive, resulting in unnatural smile animation. The recipient nerves for both of these patients were the ipsilateral facial nerve stumps, which were dissected by opening the facial nerve canal in the mastoid process. From the standpoint of operative technique, the one-stage transfer for latissimus dorsi muscle appears superior. Namely, a combined soft-tissue flap can provide sufficient augmentation for depression of the parotid region following wide resection. A long vascular stalk of thoracodorsal vessels is also useful for anastomosis, with recipient vessels available after extensive ablation and neck dissection.     

Effects of mandibular distraction osteogenesis on the inferior alveolar nerve: an experimental study in monkeys

A series of experimental studies were performed in monkeys to study the effect of mandibular distraction osteogenesis on the inferior alveolar nerve at different times before and after distraction. A mandible osteotomy was performed and distraction was carried out unilaterally in 10 young rhesus monkeys and bilaterally in six. The intact nerves on the contralateral side of the 10 monkeys were used for the control. Care was taken to avoid destroying the integrity of the inferior alveolar nerve during the surgical procedure. After a 5-day latency period, the distraction device was activated at a rate of 0.5 mm twice each day for 15 days. Sensory nerve action potential testing was applied before and 1 day after the operation, at completion of distraction, and at 2, 4, 6, 9, and 12 weeks of fixation. Necropsy was performed at the completion of distraction and 2, 4, 6, and 12 weeks of fixation. The mental nerves were taken, sectioned, and stained with lead citrate and uranyl acetate, and examined with a transmission electron microscope. The inferior alveolar nerves in the distraction gap were obtained, and paraffin slides were made and stained with hematoxylin and eosin, Luxol fast blue, and Bodian methods. The authors found that immediately after the mandible osteotomy, most nerves showed signs of slight acute injury; the latency was increased by 5.553 percent, and the amplitude was decreased to 1808 microV. This might be caused by the surgical procedure or by compressions produced by swelling tissues around the nerves. When distraction was completed, the latency was prolonged for an average of 22.18 percent, and the amplitude average had attenuated to 28.54 percent (804 microV) of the preoperative value on the distracted side. Most nerve fibers exhibited signs of degeneration, such as myelin disruption, swelling of cell organs greatly increased in axoplasm, axon tearing, and myelin fragments engulfed by macrophages. These were nerve reactions to the tensions produced by mandible lengthening. As time elapsed, the nerve's action potential recovered gradually because of its repairing ability, the latency shortened, amplitude increased, Schwann cells proliferated and formed new myelin sheaths, and the tearing axons reconnected. After 12 weeks of consolidation, there was still a latency of 12.384 percent prolongation because of the prolonged conduction distance, and the average amplitude was restored to 2786 microV, the approximate preoperative value. The nerve seemed to be repaired completely; its myelin thickness, axon diameter, and ultrastructure were all similar to those of the control. It was concluded that mandibular distraction osteogenesis can produce some degree of harmful effects on the function and structure of inferior alveolar nerves, but it is reversible and relatively slight. Along with the regeneration of the nerve's myelin and axon, the nerve function can gradually rehabilitate to a normal level.     

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4–L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.     

Role of endothelin and endothelin A-type receptor in adaptation of the carotid body to chronic hypoxia

Chronic exposure in a low-PO(2) environment (i.e., chronic hypoxia, CH) elicits an elevated hypoxic ventilatory response and increased hypoxic chemosensitivity in arterial chemoreceptors in the carotid body. In the present study, we examine the hypothesis that changes in chemosensitivity are mediated by endothelin (ET), a 21-amino-acid peptide, and ET(A) receptors, both of which are normally expressed by O(2)-sensitive type I cells. Immunocytochemical staining showed incremental increases in ET and ET(A) expression in type I cells after 3, 7, and 14 days of CH (380 Torr). Peptide and receptor upregulation was confirmed in quantitative RT-PCR assays conducted after 14 days of CH. In vitro recordings of carotid sinus nerve activity after in vivo exposure to CH for 1-16 days demonstrated a time-dependent increase in chemoreceptor activity evoked by acute hypoxia. In normal carotid body, the specific ET(A) antagonist BQ-123 (5 microM) inhibited 11% of the nerve discharge elicited by hypoxia, and after 3 days of CH the drug diminished the hypoxia-evoked discharge by 20% (P < 0.01). This inhibitory effect progressed to 45% at day 9 of CH and to nearly 50% after 12, 14, and 16 days of CH. Furthermore, in the presence of BQ-123, the magnitude of the activity evoked by hypoxia did not differ in normal vs. CH preparations, indicating that the increased activity was the result of endogenous ET acting on an increasing number of ET(A). Collectively, our data suggest that ET and ET(A) autoreceptors on O(2)-sensitive type I cells play a critical role in CH-induced increased chemosensitivity in the rat carotid body.     

Retinoic acid reverses the airway hyperresponsiveness but not the parenchymal defect that is associated with vitamin A deficiency

Airway hyperresponsiveness (AHR) is influenced by structural components of the bronchial wall, including the smooth muscle and connective tissue elements and the neuromuscular function. AHR is also influenced by parenchymally derived tethering forces on the bronchial wall, which maintain airway caliber by producing outward radial traction. Our previous work has shown that vitamin A-deficient (VAD) rats exhibit cholinergic hyperresponsiveness and a decrease in the expression and function of the muscarinic-2 receptors (M2R). We hypothesized that if decreases in radial traction from airway or parenchymal structures contributed to the VAD-related increase in AHR, then the radial traction would normalize more slowly than VAD-related alterations in neurotransmitter signaling. Rats remained vitamin A sufficient (VAS) or were rendered VAD and then maintained on the VAD diet in the presence or absence of supplementation with all-trans retinoic acid (RA). VAD was associated with an approximately twofold increase in respiratory resistance and elastance compared with VAS rats. Exposure to RA for 12 days but not 4 days restored resistance and elastance to control (VAS) levels. In VAD rats, AHR was accompanied by decreases in bronchial M2R gene expression and function, which were restored after 12 days of RA supplementation. Subepithelial bronchial elastic fibers were decreased by approximately 50% in VAD rats and were significantly restored by RA. The increase in AHR that is associated with VAD is accompanied by decreases in M2R expression and function that can be restored by RA and a reduction in airway elastic fibers that can be partially restored by RA.     

Method of combined detection of nerve fibers and blood vessels in nerve trunks]

A new method for investigation of neuro-vasal relationships in nerve conductors is proposed based on a combination of the fine injecting of their blood vessels and staining the nerve fibres. The vessels are injected with the chloroform emulsion of finely ground Paris blue (5-10 g per 100 g of solvent). Pieces of nerves 0,5 cm thick are fixed in 12% neutral formalin for 3 days, kept in a dark vessel in Weigert's mortant for 5 days, dehydrated and imbedded in paraffin. Thin slices 4-5 micronkm thick are stained in hematoxylin after N. N. Kulchitski, differentiated for 2-12 h in the mixture containing 1% solution of potassium ferricyanide and saturated solution of lithium carbonate (1:10) and after passing through alcohols of increasing concentration and xylene imbedded in balsam. In the preparations the fibres of different caliber are stained grey and the vessels are stained blue.     

Changes of nitric oxide synthase-containing nerve fibers and parameters for oxidative stress after unilateral cavernous nerve resection or manuplation in rat penis

After pelvic surgeries such as radical prostatectomy, two major complications--urinary incontinence and erectile dysfunction (ED) may occur. Etiologies for ED are multiple pathologic mediators/systems. Oxidative stress, which is known to be induced after surgical trauma, could be a cause of ED. The purposes of in this study are to investigate the effect of unilateral manipulation/ dissection and resection of the cavernous nerve (neurotomy) to NOS (nitric oxide synthase)-containing nerve fibers and pressure after electro stimulation in rat corpus cavernosum, and to determine whether these procedures would produce oxidative stress within rat cavernous tissue 3 weeks and 6 months after the operation. Male rats were divided into 5 groups. Rats in groups 1 and 2 underwent unilateral cavernous nerve manipulation and sacrificed 3 weeks and 6 months after the operation, respectively. Rats in groups 3 and 4 underwent unilateral neurotomy of a 5-mm. segment of the cavernous nerve, and they were sacrificed 3 weeks and 6 months after nerve ablation, respectively. Group 5 rats were control animals for biochemical analysis. Intracavernous pressure following electro stimulation reduced is significantly 3 weeks after unilateral resection, as compared to that of the manipulated nerve (P < 0.05), and it recovered 6 months after neurotomy. The recovery was also confirmed by NADPH (nicotinamide adenine dinucleotide phosphate) diaphorase staining in neurotomy groups. Lipid peroxidation, which is an indicater of oxidative stress, was determined by measuring thiobarbituric acid reacting substance (TBARS) levels and superoxide dismutase (SOD) activity. These markers indicated that unilateral cavernous nerve manipulation or resection produced oxidative stress within rat corpus cavernosum. Oxidative stress was more prominent 3 weeks after unilateral neurotomy (P < 0.05). Also, compared to the control animal group, oxidative stress was observed three weeks after manipulation of unilateral cavernous nerve (P < 0.05). Resection of the cavernous nerve caused more prominent oxidative stress than in the manipulation group. This study suggested, that unilateral cavernous neurotomy caused a decrease of intra cavernous pressure and NOS fibers in rat corpus cavernosum, and they recovered 6 months after neurotomy. Our data also provided evidence that neurotomy and manipulation of the cavernous nerve caused oxidative stress in rat corpus cavernosum and that oxidative stress was more prominent in the nerve resection group.     

Traction avulsion amputation of the major upper limb: a proposed new classification, guidelines for acute management, and strategies for secondary reconstruction.

Major replantation of a traction avulsion amputation is undertaken with the goal of not only the reestablishment of circulation, but also functional outcome. This type of amputation is characterized by different levels of soft-tissue divisions involving crushing, traction, and avulsion injuries to various structures. Between 1985 and 1998, 27 cases were referred for secondary reconstruction following amputation of the upper extremity involving both arm and forearm. Replantation was performed by at least 12 qualified plastic surgeons using different approaches and management, resulting in different outcomes. Initial replantation management significantly affects the later reconstruction. For comparing studies and prognostic implications, the authors propose a new classification according to the level of injury to muscles and innervated nerves: type I, amputation at or close to the musculotendinous aponeurosis with muscles remaining essentially intact; type II, amputation within the muscle bellies but with the proximal muscles still innervated; type III, amputation involving the motor nerve or neuromuscular junction, thereby causing total loss of muscle function; and type IV, amputation through the joint; i.e., disarticulation of the elbow or shoulder joint. Some patients required further reconstruction for functional restoration after replantation, but some did not. Through this retrospective study based on the proposed classification system, prospective guidelines for the management of different types of traction avulsion amputation are provided, including the value of replantation, length of bone shortening, primary or delayed muscle or nerve repair, necessity of fasciotomy, timing for using free tissue transfer for wound coverage, and the role of functioning free muscle transplantation for late reconstruction. The final functional outcome can also be anticipated prospectively through this classification system.     

Prelaminating the fascial radial forearm flap by using tissue-engineered mucosa: improvement of donor and recipient sites.

In reconstructive surgery, prelamination of free flaps using split-thickness skin is an established technique to avoid the creation of a considerable defect at the donor site, for example, in the case of a radial forearm flap. For oral and maxillofacial surgery, this technique is less than optimal for the recipient site because the transferred skin is inadequate to form a lining in the oral cavity. To create mucosa-lined free flaps, prelamination using pieces of split-thickness mucosa has been performed. However, the availability of donor sites for harvesting mucosa is limited. The present study combines a tissue-engineering technique with free flap surgery to create mucosa-lined flaps with the intention of improving the tissue quality at the recipient site and decreasing donor-site morbidity. On five patients undergoing resection of squamous cell carcinoma of the oral cavity, the radial forearm flap was prelaminated with a tissue-engineered mucosa graft to reconstruct intraoral defects. Using 10 x 5 mm biopsies of healthy mucosa, keratinocytes were cultured for 12 days and seeded onto collagen membranes (4.5 x 9 cm). After 3 days, the mucosal keratinocyte collagen membrane was implanted subcutaneously at the left or right lower forearm to prelaminate the fascial radial forearm flap. One week later, resection of the squamous cell carcinoma was performed, and the free fascial radial forearm flap pre- laminated with tissue-engineered mucosa was transplanted into the defect and was microvascularly anastomosed. Resection defects up to a size of 5 x 8 cm were covered. In four patients, the graft healed without complications. In one patient, an abscess developed in the resection cavity without jeopardizing the flap. During the postoperative healing period, the membrane detached and a vulnerable pale-pink, glassy hyperproliferative wound surface was observed. This surface developed into normal-appearing healthy mucosa after 3 to 4 weeks. In the postoperative follow-up period, such functions as mouth opening and closing and speech attested to the success of the tissue-engineering technique for flap prelamination.