Carbonic anhydrase histochemistry. A potential diagnostic method for peripheral nerve repair

Many large-diameter myelinated axons in spinal dorsal roots contain carbonic anhydrase activity, whereas few small-diameter ventral root axons stain for this enzyme. This differential localization of carbonic anhydrase in sensory and motor nerve fibers is indicative of the potential of carbonic anhydrase histochemistry to provide a convenient method for identifying predominantly motor or sensory fascicles in cut ends of peripheral nerves, thereby facilitating coaptation of fascicles in peripheral nerve repair.     

Histochemical staining of nerve endings as an aid to free muscle transplantation.

Histochemical staining techniques that identify intact motor nerve fascicles are available to aid free muscle transplantation. Cholinesterase activity of myelinated axons can be identified by Karnovsky and Roots's technique. Axon viability can be assessed based on the presence of axoplasmic enzyme activity. By reacting serial sections for cholinesterase activity and carbonic anhydrase activity, which labels sensory axons, an accurate cross-sectional map of regenerating or functional sensory and motor nerve fibers can be constructed. Resolving the motor and sensory identities of fascicles in a mixed peripheral nerve should lead to more precise coaptation of recipient motor fibers to the motor nerve of the transferred muscle and enhance reinnervation.     

Identification of myelinated motor and sensory axons in a regenerating mixed nerve

The common peroneal nerves of Wistar rats were transected and repaired to compare the sequential changes in the numbers of regenerating motor and sensory myelinated axons in a single mixed nerve. At sequential intervals (2, 4, and 12 weeks) after nerve repair, 3 kinds of staining were performed: cholinesterase staining (Karnovsky's staining) for motor axons, carbonic anhydrase staining for sensory axons, and antineurofilament immunohistochemical staining for all axons. At 2 weeks there was a large number of carbonic anhydrase-positive axons (600 +/- 98; mean +/- SD) and cholinesterase-positive axons were occasionally seen. Subsequently, there was a striking increase of cholinesterase-positive myelinated axons, reaching to 302 +/- 50 at 12 weeks. The results suggest that the myelinated sensory axons regenerate faster in the early stage of nerve regeneration and that regeneration of the myelinated motor axons is prominent in the subsequent stage. (J Hand Surg 2000; 25A:104-111.     

Histochemical discrimination of fibers in regenerating rat infraorbital nerve.

In rat dorsal root ganglia, histochemical staining of carbonic anhydrase (CA) and cholinesterase (CE) yields a reciprocal pattern of activity: Sensory processes are CA positive and CE negative, whereas motor processes are CA negative and CE positive. In rat infraorbital nerve (a sensory peripheral nerve), we saw extensive CA staining of nearly 100% of the myelinated axons. Although CE reactivity in myelinated axons was extremely rare, we did observe CE staining of unmyelinated autonomic fibers. Four weeks after transection of infraorbital nerves, CA-stained longitudinal sections of the proximal stump demonstrated 3 distinct morphological zones. A fraction of the viable axons retained CA activity to within 2 mm of the distal extent of the stump, and the stain is capable of resolving growth sprouts being regenerated from these fibers. Staining of unmyelinated autonomic fibers in serial sections shows that CE activity was not retained as far distally as is the CA sensory staining.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

人源性同种异体去细胞外周神经材料标准制备方法的研究

目的 对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣,以确定可供临床使用的标准的制备流程.方法 将人源性神经按5种不同方法进行化学萃取,制备的外周神经支架材料分别行苏木精-伊红(HE)染色、免疫组化(S-100、Col I)和透射电镜、氮含量测定等检测,观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况.结果 分别萃取2次持续24 h组神经的HE染色显示去除细胞和轴突彻底,纵切片上未见任何细胞,红染的神经内膜呈波浪状纵形排列,轴突、髓鞘结构消失而形成管柱状空隙;S-100染色呈阴性;Col I染色结果可以看出其结构呈松散不规则的棕黄色结构,而其它处理组则结构相对整齐的纵向带状结构.透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义.氮含量测定显示该组蛋白含量比值最低.结论 使用Triton X-100处理24 h再分别脱氧胆酸钠24 h持续萃取2次,可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程.     

Motor evoked potentials enable differentiation between motor and sensory branches of peripheral nerves in animal experiments

Differentiation between motor and sensory fascicles is frequently necessary in reconstructive peripheral nerve surgery. The goal of this experimental study was to verify if centrally motor evoked potentials (MEP) could be implemented to differentiate sensory from motor fascicles, despite the well-known intermingling between nerve fascicles along their course to their distant periphery. This new procedure would enable surgeons to use MEP for placing nerve grafts at corresponding fascicles in the proximal and distal stumps without the need to use time-consuming staining. In ten sheep, both ulnar nerves were exposed at the terminal bifurcation between the last sensory and motor branch. Animals were then relaxed to avoid volume conduction. On central stimulation, the evoked nerve compound action potentials were simultaneously recorded from both terminal branches. In all cases, neurogenic motor nerve action potentials were recorded only from the terminal motor branch. The conclusion was that MEPs can be used for intraoperative differentiation between sensory and motor nerves. Further studies are necessary to develop this method for in situ measurements on intact nerve trunks.     

Reinnervation of muscles in rats after repair of transsected sciatic nerves with Y-shaped and X-shaped silicone tubes. Muscle reinnervation after nerve repair.

Reinnervation of the gastrocnemius and anterior tibial muscles was assessed by measurements of tetanic force after repair of sciatic nerves with Y-shaped or X-shaped silicone tubes in rats. The transsected proximal stump of either the tibial or the peroneal fascicle was introduced into the opening of a Y-shaped silicone tube, or both fascicles were introduced into an X-shaped tube. The distal tibial and peroneal fascicles were inserted into the distal outlets of the tubes leaving a gap of 4 mm between proximal and distal stumps. In the X-shaped tubes the proximal inserts were placed opposite or adjacent to their respective distal parts. Sixteen weeks later reinnervation was evaluated by measurements of tetanic force of the gastrocnemius and anterior tibial muscles after electrical stimulation of the fascicles. There was preferential reinnervation in both types of tubes. In Y-shaped tubes about 90% of the tetanic force could be recorded from both muscles after stimulation of the peroneal and tibial fascicles, respectively. Recovery was lower in the X-shaped tubes, amounting to about 75%. Contractions evoked by misrouted fibres were similar (roughly 40%) in both models. We conclude that motor axons preferentially, but not exclusively, selected a path to reinnervate their original target muscle.     

Sensory and motor fiber differentiation with Karnovsky staining.

We examined four acetylthiocholine methods based on Karnovsky's procedure--two fast-acting requiring 1 hour and two slow-acting requiring 24 hours. We compared these with our modification, which requires less than an hour and is simple to use. Rabbit sciatic nerves and spinal cords were used to compare methods. Our modification showed clearer differentiation than other fast-acting methods and staining identical to slow-acting methods. In blind examination of radial nerve specimens stained with our method, motor and sensory fascicles were correctly identified, showing sensitivity and specificity of 100%. In 12 clinical cases, our method produced staining in the proximal stump as long as 16 months after injury and in the distal stump as long as 5 days after injury. In 10 of 12 patients, this staining helped in aligning motor fascicles to motor fascicles and sensory fascicles to sensory fascicles.     

Cranial root injury in glossopharyngeal neuralgia: electron microscopic observations. Case report

Optical and electron microscopic examinations were made of a biopsy sample of the ninth and 10th cranial nerves obtained during posterior fossa surgery for the relief of pain in a patient suffering from glossopharyngeal neuralgia (GN). Pathological findings, which were restricted to a small fraction of fascicles in the nerves, included large patches of demyelinated axons in close membrane-to-membrane apposition to one another and zones of less severe myelin damage (dysmyelination). These observations, in the light of similar morphological changes observed in biopsy samples excised from patients with trigeminal neuralgia, and new information on the pathophysiological characteristics of injured peripheral nerve axons, can account for much of the symptomatology of GN.     

Olfaction preservation in anterior cranial base approaches: an anatomic study

OBJECTIVE: To study the anatomic basis for olfaction-sparing anterior cranial base approaches. METHODS: The medial anterior skull base containing the olfactory unit and delimited by the inner table of the frontal sinus, the lesser wing of the sphenoid bone, and the medial orbital walls was removed from six cadaveric specimens. Histological methods were used to investigate the location, distribution, and depth of penetration of olfactory nerves. Hematoxylin and eosin and Gomori trichrome staining were used to visualize landmarks and architecture. S-100 neurofilament protein immunostaining was used to identify nerve fascicles and axons. In three cadaveric head specimens, olfaction-sparing craniofacial approaches were performed and the excised olfactory units were evaluated histologically. RESULTS: Bundles of olfactory nerves were identified primarily in the nasal septum; relatively fewer bundles could be identified in the middle turbinate. Olfactory nerve endings were identified up to 20 mm below the cribriform plate (range, 7-20 mm). The superior and middle nasal meatus were most innervated; olfactory innervation was virtually absent in the inferior nasal meatus. Histological evaluation of the olfactory unit elevated during olfaction-sparing techniques routinely revealed transection of olfactory nerves that exited the skull base. CONCLUSION: In olfaction-sparing anterior cranial base approaches, the olfactory nerves are inevitably transected. The clinical significance of olfactory nerve transection for postoperative functional recovery of olfaction remains to be analyzed.