PC12 cell aggregation and neurite growth in gels of collagen, laminin and fibronectin

PC12 cells form aggregates when suspended within three-dimensional, self-assembled, type I collagen gels; these aggregates increase in size over time. In addition, when the cells are cultured in the presence of nerve growth factor, they express neurites, which extend through the three-dimensional matrix. In this report, the roles of fibronectin, laminin and nerve growth factor in PC12 cell aggregation and neurite growth following suspension in collagen matrices were evaluated. Single cells and small clusters of cells were suspended in collagen gels; the kinetics of aggregation were determined by measurement of the projected area of each aggregate, and neurite lengths were determined by measurement of end-to-end distance. Fibronectin and laminin inhibited the aggregation of PC12 cells at 50 micrograms/ml, and fibronectin, but not laminin, inhibited the growth of neurites at 100 micrograms/ml. In the absence of serum, the aggregation of cells cultured with nerve growth factor was almost completely inhibited, but the average neurite length was unaffected. In the presence of nerve growth factor, the extent of cell aggregation could not be explained simply by an increase in cell number, suggesting the presence of two separate mechanisms for aggregate growth: one dependent on cell motility and another dependent on cell proliferation.     

An electron microscopic study of the processes of nerve regeneration after lyophilized nerve homografting

The objective of the present study was to investigate the role of the Schwann cell basal lamina in nerve regeneration. To achieve this goal, we observed the process of axonal regeneration within a lyophilized nerve graft, in which only the basal lamina of the Schwann cell persisted. Sciatic nerves were removed from rats and lyophilized to kill the Schwann cells and other components. These grafts were transplanted to rat sciatic nerve defects. The rats were then killed after lapses of time. We observed the processes of axonal regeneration using a transmission electron microscope. Regeneration of axons along the inner surface of the Schwann cell basal lamina was clearly seen. These results suggest that, if tubular basal laminae persist, Schwann cells are not always necessary, and axonal regeneration can be induced in the direction toward the basal lamina.     

Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.     

Fracture healing and callus innervation after peripheral nerve resection in rats

The effects of femoral and sciatic nerve resection on fracture healing and innervation of the fracture callus were studied using a stable fracture model. In 34 rats the right tibia was subjected to a standardized closed fracture and stabilized with a modular intramedullary nail. In half of the animals, resection of 1 cm of the femoral and sciatic nerves was performed (nerve resection group), whereas the other animals had sham operations (sham group). To avoid unequal load-bearing between the two groups, all fractured hindlimbs were immobilized in a plaster of Paris cast. The trial was terminated after 5 weeks of fracture healing. Callus size was scored radiographically, and bone mineralization was measured by 85-strontium incorporation. Seven rats from each group had immunohistochemical examination for neural regeneration and ingrowth. Antisera for protein gene product 9.5, neurofilaments, neural growth associated protein 43/B-50, calcitonin gene related peptide, and substance P were used. The mechanical properties of the healing fractures were recorded in a three-point cantilever bending test. After 5 weeks, the normally innervated, fractured tibias had regained approximately 50% strength compared with the unfractured side, in comparison with only 20% in the animals that had nerve resection. Although the fracture calluses were mechanically weaker, they were significantly larger in the nerve resection group, indicating defects in tissue composition or organization rendered by the nerve injury. The mineralization rate, as measured by 85-strontium incorporation, was the same in the two groups. However, the nerve resection did not provide complete denervation but changed the innervation pattern of the healing fracture, as the density of sensory nerve fibers immunostaining for substance P and neurofilaments was less in the group with femoral and sciatic nerve resection. The results suggest that intact innervation is essential for normal fracture healing because nerve injury induced a large, but mechanically insufficient, fracture callus.     

Fatigue analysis of human reinnervated muscle after microsurgical nerve repair

The reinnervated elbow flexors, biceps, and brachialis muscles were compared with the elbow flexors on the healthy opposite side in terms of muscle strength and fatigue in 10 patients who sustained sequelae of a unilateral posttraumatic brachial plexus palsy. The patients had recovered an active elbow flexion against resistance after microsurgical nerve repair. The patients were reviewed with an average postoperative followup of 12 years (range, 7.5-16 years). Despite a statistically significant difference in maximum isometric force, this study showed that after peripheral nerve repair, a partially reinnervated muscle has the same characteristics of fatigue and endurance as a normally innervated muscle, if these muscles exert the same percentage of their own maximum force.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Intraoperative facial nerve monitoring (IFNM) predicts facial nerve outcome after resection of vestibular schwannoma

Intraoperative facial nerve monitoring (IFNM) is a suitable technique for intraoperative facial nerve identification and dissection, especially in large vestibular schwannomas (VS) (acoustic neuroma). To evaluate its feasibility for estimating functional nerve outcome after VS resection 60 patients underwent surgery using IFNM. Out of this group the last 40 patients were included in a prospective study evaluating the prognostic value of various IFNM parameters (proximal and distal absolute EMG amplitude, stimulation threshold, and proximal-to-distal amplitude ratio) for prediction of initial postoperative facial nerve function and recovery of function. Stimulation threshold and absolute EMG amplitude proximally at the brain stem were both predictive for postoperative nerve function. Good initial facial nerve outcome (modified House Brackmann grading, mHB degree I and degree II) was found in 15/16 patients with a proximal EMG amplitude greater than 800 microV and in 19/22 patients with proximal stimulation threshold less than 0.3 mA. Sixteen of 16 patients with proximal stimulation threshold equal to or greater than 0.3 mA had moderate-to-severe facial palsy (mHB degree III or worse). Six of six patients without evokable proximal amplitude initially had insufficient nerve function (mHB degree IV). Intraoperative decrease of the proximal amplitude was associated with an unfavourable outcome, whereas distal amplitudes usually stayed unchanged. Mean distal EMG amplitudes were also found to be decreased with poor nerve function, which may mean that the tumour had already affected the nerve. A proximal amplitude of 300 microV or less and a proximal-to-distal amplitude ratio below 1:3 were found in the absence of functional recovery in 6/8 (75%) and 5/6 (83%) patients with initial mHB degree IV, respectively. Two patients with initial mHB degree IV improved to mHB degree III despite intraoperative evidence of missing functional nerve integrity. Therefore, functional recovery cannot be predicted by IFNM in all cases of anatomical nerve preservation. We conclude that a minimum follow-up period of 1 year may still be advisable even in certain patients without evidence of intraoperative functional nerve integrity.     

Effect of amniotic fluid in corneal sensitivity and nerve regeneration after excimer laser ablation

To examine the effect of topical application amniotic fluids on the recovery of corneal sensitivity and nerve regeneration after excimer laser photokeratectomy, excimer laser was applied to 18 rabbits (VISX 20/20, 5 Hz, 7 microns depth: nine rabbits; 100 microns depth: nine rabbits). Human amniotic fluid (AF) was topically applied to the right eyes (AF group), and a balanced salt solution (BSS) was applied to the left eyes (BSS group). Corneal sensitivity was measured by using a Cochet-Bonnet aesthesiometer after weeks 1, 2, 4, 5, 8, and 12. Nerve-regeneration status was evaluated after weeks 2, 4, 5, 8, and 12 by gold chloride staining. Corneal sensitivity was initially subnormal and recovered close to the normal level at week 8. The sensitivity was higher in the AF group than in the BSS control group, except at week 2. Subepithelial nerve regeneration at the laser site was detected both at week 4 in the AF group and at week 5 in the BSS group. There was no significant correlation between the nerve-regeneration state and sensitivity changes. Long striated nerve regeneration from the deep stromal nerve was remarkable at the third month, especially in the BSS group. The BSS group showed more marked scarring of the superficial stroma, compared with the AF group. The nerve regeneration at the scar site was discontinued and delayed. Subepithelial leash nerves in the AF group were more abundant than those in the BSS group. The recovery of sensitivity and nerve regeneration were faster in the AF cornea than in the BSS cornea. These results suggest that the factors in AF helped the recovery of corneal sensitivity, nerve regeneration, and reduced scar formation.     

Abduction defect associated with aberrant regeneration of the oculomotor nerve after intracranial aneurysm

PURPOSE: To determine the cause of delayed-onset ipsilateral abduction defect associated with aberrant regeneration of the oculomotor nerve. METHODS: Isolated oculomotor palsy was noted after successful basilar artery aneurysm surgery in a 35-year-old patient. Several months later, aberrant regeneration of the oculomotor nerve and an ipsilateral abduction defect were first detected. RESULTS: Ocular electromyography demonstrated failure of relaxation of the ipsilateral medial rectus muscle on attempted abduction, suggesting cocontraction of horizontal recti muscles as the origin of the abduction defect. CONCLUSION: A late-onset ipsilateral abduction defect caused by failure of relaxation of the medial rectus muscle may be associated with basilar aneurysm.     

Postprandial serum concentration of individual bile acids in man. Influence of ileal resection

For the purpose of characterizing the importance of intestinal function in the maintenance of the enterohepatic circulation of individual bile acids, the serum concentrations of cholic acid (C), chenodeoxycholic acid (CD), and deoxycholic acid (D) were determined after a standardized meal. Two groups of subjects were included: 10 healthy controls and 7 patients with ileal resections 20-80 cm long. The bile acid concentrations were determined using a highly specific and accurate gas chromatographic-mass spectrometric technique with the aid of deuterium-labelled internal standards. In patients with ileal resections, only a moderate and early increase was seen in C concentration after a meal, whereas the CD elevation was more pronounced and prolonged. The D concentrations were reduced both after fasting and postprandially. The data indicate that ileal resection results in essentially complete absence of active bile acid resorption and that intestinal uptake via nonionic diffusion is probably dominant, resulting in postprandial rise of mainly CD in serum.     

解析参数对活性焦再生过程及再生效果的影响

 活性焦的热解析参数对再生活性焦的脱硫脱硝性能和机械强度至关重要。为了明确解析参数对活性焦再生过程和再生效果的影响规律,通过热解析试验探究活性焦硫残余比例、CO₂和CO生成量及再生活性焦脱硫脱硝性能随解析温度和解析时间的变化规律,继而明确适宜的活性焦热解析参数。结果表明,活性焦升温解析过程中,脱硫产物在317 ℃左右迅速分解,随后分解速率下降;在进入恒温解析阶段后脱硫产物分解速率先快速下降,而后进入缓慢解析状态。硫残余比例随恒温解析温度的升高而下降,在530 ℃下解析3 h可使脱硫产物完全解析;解析温度高于430 ℃后,活性焦表面的酚基、醌基、内酯基等含氧官能团分解量明显增加,并随恒温解析温度的升高而持续增加,分解所生成的CO和CO₂也随之大幅增加,这将使活性焦的孔隙结构进一步发展,继而不利于活性焦机械强度的保持;解析温度低于530 ℃时,硫残余比例随解析温度的升高而持续降低,使再生活性焦的脱硫脱硝性能持续提高;解析温度高于530 ℃后,含氧官能团分解量随解析温度的升高而持续增加,这将有利于提高活性焦表面SO₂氧化反应速率,继而使再生活性焦的脱硫性能持续升高,但酚基、内酯基等酸性含氧官能团的分解使再生活性焦对NH₃的吸附性能降低,进而使其脱硝性能降低。在兼顾再生活性焦脱硫脱硝性能、机械强度和生产效率等多方面因素时,430 ℃恒温解析3 h是相对较优的解析参数。在此解析条件下,再生活性焦的硫残余比例仅为1.8%,含氧官能团尚未发生大量分解,脱硫脱硝性能相对较为优良。     

Capability of neurite regeneration of retinal explant from adult rat after optic nerve injury

Axons of the central nervous system in adult mammals do not regenerate spontaneously after axotomy. To understand whether the optic nerve of adult mammals loses the intrinsic capability to regenerate after injury, we have studied the capability of neurite regeneration of retinal explant from adult rat after optic nerve axotomy in vitro. After experimental blunt damage to the optic nerve of adult Wistar rat, the retinal explants from three days, one week, two weeks and three weeks after axotomy were put in tissue culture to observe the neurite growth after four days' incubation. The neurites were identified as retinal neuron origin by immunocytochemical staining using monoclonal antibody to neurofilament. The results demonstrate that retinal explants from adult rat after optic nerve damage have the capability of neurite regeneration; the capability is strongest in the group of one week after axotomy of optic nerve, but it decreases with passage of the time. On the other hand, the retinal explant from the control group of uninjured eye does not regenerate neurite in tissue culture. These results indicate that the retinal explant of adult rat has intrinsic capability to regenerate after optic nerve injury in vitro, and the capability of neurite regeneration decreases after one week post-trauma.     

Full functional recovery after surgical repair of transected abducens nerve: case report

OBJECTIVE AND IMPORTANCE: Results of surgical repair of the injured abducens nerve are rarely reported in the literature. A full functional recovery of a completely resected abducens root may be exceptional. We describe a patient who obtained normal ocular alignment and binocular vision after surgical reconstruction of a transected abducens nerve. CLINICAL PRESENTATION: A 56-year-old woman with a petroclival meningioma was presented. She underwent total removal of the tumor through a combined supra/infratentorial transpetrosal approach. The abducens nerve was tightly attenuated by the tumor and thickened dura. During dissection, the nerve was completely transected just behind the entrance to Dorello's canal. INTERVENTION: The abducens nerve was the single root type and inevitably required surgical repair. To obtain a sufficient length of the distal stump for trimming, part of the petrosphenoidal ligament was cut and the superior border of the petrous bone was exposed. The proximal stump of the nerve was also trimmed to obtain healthy tissue, and reconstruction was performed with five 10-0 nylon sutures. Five months later, esodeviation began to improve. Nine months after the surgery, the patient did not complain of diplopia and an objective assessment reported normal ocular alignment and estimated binocular function as "excellent" according to Biglan's system. Overcorrection of abduction did not occur. CONCLUSION: The result in our patient confirms the possibility of full functional recovery after surgical repair of a totally transected abducens nerve.     

An experimental study on the recovery of the lingual nerve after injury with or without repair

BACKGROUND: The advantage of radionuclide angiographic techniques used to measure right ventricular ejection fraction (RVEF) is geometry independence, but the weakness is right atrial (RA) overlap. To minimize the effect of RA counts on right ventricular time activity curve (TAC), two regions of interest (ROI), one drawn for the end-diastolic image and one for the end-systolic image, are used for the calculation of RVEF from equilibrium gated blood pool scans (GBPS) and from gated first-pass studies with an Anger camera. A multicrystal camera offers both temporal separation of the bolus to the right side of the heart and good count statistics; therefore first-pass studies performed on a multicrystal camera theoretically should yield the most accurate measurements of RVEF, but few studies have been performed to validate RVEF against a reliable gold standard. METHODS AND RESULTS: To develop and validate an accurate method to measure RVEF from multicrystal first-pass data, 25 patients underwent sequential cine-MRI, first-pass radionuclide angiography, and gated equilibrium imaging. Five additional healthy volunteers underwent cine-MRI alone. Right and left ventricular volumes were measured from serial short axis cine-MRI views according to Simpson's rule. Three methods were used to calculate RVEF from first-pass data: a single ROI method, a dual ROI method, and a method in which a single ROI is applied to RA subtracted first-pass dynamic data. Five additional healthy volunteers underwent cine-MRI alone. When right ventricular stroke volume was plotted versus left ventricular stroke volume for the 5 volunteers and the 15 patients without valvular regurgitation, the regression line was not significantly different from the line of identity, supporting the accuracy of cine-MRI to measure RVEF. The RVEF by cine-MRI ranged from 34% to 59%; first-pass RVEF with a single ROI from 26% to 48%; first-pass RVEF with two ROIs from 31% to 59%; first-pass RVEF with RA subtracted single ROI from 29% to 60%; and RVEF from GBPS with multiple ROIs from 28% to 55%. The regressions for all three of the first-pass methods versus cine-MRI were significant (p < 0.01) as was the regression for the equilibrium GBPS versus cine-MRI but the correlation was weaker. The regressions for the 2-ROI method and for the RA subtracted single ROI method were not significantly different from the line of identity, whereas the regressions for both the single ROI method and for equilibrium GBPS were significantly different from the line of identity (p < 0.01). CONCLUSIONS: Cine-MRI can be used to validate radionuclide algorithms. Of the four radionuclide methods for measuring RVEF that were assessed, the first-pass 2-ROI method and the first-pass RA subtracted single ROI are the most accurate, the first-pass single ROI method underestimates RVEF, and the RVEF values measured from GBPS are less accurate.     

Phenotypic modulation and elastin formation of cultured aortic smooth muscle cells grown on and within collagen gels

Aortic smooth muscle cells (SMC) were grown on and within collagen gels and their phenotype and elastin formation were examined. Cells grown on the gels showed multilayer growth with nodule formation. They contained many ribosomes and microfilament bundles with dense bodies. In contrast, cells grown within collagen gels did not form nodules. When collagen gels containing SMC were manually detached from the culture dish, the cells were found to be of the synthetic phenotype with well-developed rough endoplasmic reticulum (ER) and Golgi complexes, but few microfilament bundles. There were numerous elastin deposits in the intercellular spaces. Our findings suggest that the physical property of the substrate affects the phenotype and function of the SMC.     

The cellular and molecular basis of peripheral nerve regeneration

Functional recovery from peripheral nerve injury and repair depends on a multitude of factors, both intrinsic and extrinsic to neurons. Neuronal survival after axotomy is a prerequisite for regeneration and is facilitated by an array of trophic factors from multiple sources, including neurotrophins, neuropoietic cytokines, insulin-like growth factors (IGFs), and glial-cell-line-derived neurotrophic factors (GDNFs). Axotomized neurons must switch from a transmitting mode to a growth mode and express growth-associated proteins, such as GAP-43, tubulin, and actin, as well as an array of novel neuropeptides and cytokines, all of which have the potential to promote axonal regeneration. Axonal sprouts must reach the distal nerve stump at a time when its growth support is optimal. Schwann cells in the distal stump undergo proliferation and phenotypical changes to prepare the local environment to be favorable for axonal regeneration. Schwann cells play an indispensable role in promoting regeneration by increasing their synthesis of surface cell adhesion molecules (CAMs), such as N-CAM, Ng-CAM/L1, N-cadherin, and L2/HNK-1, by elaborating basement membrane that contains many extracellular matrix proteins, such as laminin, fibronectin, and tenascin, and by producing many neurotrophic factors and their receptors. However, the growth support provided by the distal nerve stump and the capacity of the axotomized neurons to regenerate axons may not be sustained indefinitely. Axonal regenerations may be facilitated by new strategies that enhance the growth potential of neurons and optimize the growth support of the distal nerve stump in combination with prompt nerve repair.     

Distribution of laminin, type IV collagen and fibronectin in the invasive component of breast carcinoma

Laminin, type IV collagen and fibronectin were examined immunohistochemically in the invasive component of breast carcinomas. Laminin was expressed around the invasive carcinoma cell nests in 38 (54%) of 71 cases. Immunoreactivity for type IV collagen was observed around the invasive carcinoma cell nests or the stroma apart from carcinoma cells in 44 (80%) of 55 cases. Fibronectin was strongly expressed in the stroma only in 75 (99%) of 76 cases. The expression of laminin significantly correlated with tubular formation in the invasive carcinoma cell nests and showed a tendency to be correlative to estrogen receptor (ER) and progesterone receptor (PgR) of carcinoma tissue, but no correlation among laminin expression, histological type, the age of patients, tumor size and lymph node metastasis was noted. Type IV collagen and fibronectin did not correlate to any clinicopathological factors such as histological type, grade of differentiation, the age of patients, tumor size, lymph node metastasis, ER and PgR status. No concordant expression of these extracellular matrices was seen.