Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 59, Num. 3, 2011, pp. 333-338

Neurology India, Vol. 59, No. 3, May-June, 2011, pp. 333-338

Original Article

Differentiation between the motor and sensory fascicles of the peripheral nerves from adult rats using annexin V-CdTe-conjugated polymer

Xianyu Meng¹, Laijin Lu², Hu Wang³, Bin Liu²

¹ Department of Orthopedics, The First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Heilongjiang; The Forth Affiliated Hospital, Harbin Medical University, Heilongjiang, China
² Department of Hand Surgery, The First Hospital, Jilin University, Changchun, China
³ Jilin Province People Hospital, Changchun, China

Correspondence Address: Laijin Lu Department of Hand Surgery, The First Hospital, Jilin University, Changchun - 130 021 China 834391482@qq.com

Date of Submission: 02-Sep-2010
Date of Decision: 30-Nov-2010
Date of Acceptance: 16-Mar-2011

Code Number: ni11105

PMID: 21743158

DOI: 10.4103/0028-3886.82710

Abstract

Keywords: Annexin V, CdTe, differentiate, peripheral nerve fascicles, quantum dots

Introduction

Peripheral nerve injury results in significant functional disability and psychological stress. Surgical approaches such as fascicular repair under a head-mounted microscope, ^[1] have greatly improved peripheral nerve repair in recent years. However, failures in nerve repair is frequent, partly because of the difficulty in differentiation between nerve fascicles. ^[2] It is thus essential to identify motor and sensory fascicles in peripheral nerve reconstructive surgery. Various approaches such as intraneural topography, ^[3],[4] electrophysiological monitoring, ^[5],[6] and enzymatic staining ^{[7],[8],[9],[10],[11],[12],[13]} have been used to address this issue, however, these approaches are imprecise, time consuming, or unsuitable for intraoperative use. Till date there has been no method available to rapidly differentiate between motor and sensory nerve fascicles intraoperatively. The identification of motor and sensory nerve fibers can help surgeons to optimally place fascicular sutures and thus the best nerve function recovery. ^[14]

Identification of a protein specific to the sensory nerves for use as a molecular marker is a promising method to identify sensory nerves in the peripheral nerves. A sensory neuronal specific protein with a molecular weight of 35 kDa has been isolated and purified from rat spinal ganglia and sensory fibers. Electrophoresis analysis showed that the protein is present in spinal sensory ganglia but not in spinal motor neurons, and it contains a mercapto group. ^[15] Results from combined direct trypsin digestion and liquid chromatography ion trap mass spectrometry analysis revealed that this 35 kDa protein was Annexin V. ^[16] These observations are consistent with the earlier reports showing the tissue distribution of Annexin V in the rat nervous system. ^[17] Annexin V was first extracted from the cholinergic nerve terminals in electric fish and was named by Walker et al. in 1982. ^[18] It is a calcium-binding protein with a molecular weight of 32-35 kDa and is a member of the Annexin family.^[19] Annexin V plays important roles in anticoagulant activity, ^[20] anti-inflammatory actions, ^[21] and calcium (Ca ²⁺ ) ion channel activity. ^[22] Recently, it was fused to green fluorescence protein (GFP-Annexin) to monitor calcium-dependent translocation. ^[23]

Quantum dots (QDs) have been covalently linked to bio-recognition molecules such as peptides, antibodies, nucleic acids, or small-molecule ligands to be used as fluorescent probes. ^{[24],[25],[26],[27],[28],[29],[30]} Antibody conjugated-QDs allow real-time imaging and tracking of a single receptor molecule on the surface of living cells with high sensitivity and resolution. ^[31] In addition, the size-dependent optical properties of QDs make them ideal candidates for tunable absorbers and emitters in various applications ranging from nanoscale electronics to biological fluorescent labeling. ^[32] Since cadmium telluride (CdTe) also plays an important role in imaging and in the manufacture of chemical sensors and biological labels, CdTe QDs have attracted attention as an optical material. ^[33] However, their applications in the identification of nerve fascicles remain unexplored. Since sensory and motor nerve fascicles intermingle with each other along the distant peripheral nerves, ^[34] we hypothesized that the application of CdTe would be an ideal way to identify the two types of nerve fascicles. In this study, our aim was to determine whether Annexin V-CdTe-conjugated polymer can be used to differentiate sensory from the motor fascicles within the peripheral nerves.

Material and Methods

Chemicals and equipment

The chemicals and equipment used in this study included: Sheep anti-rat Annexin V antibody (Santa Cruz biotechnology, CA, USA); CdTe QDs (Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, P. R. China); Leica cryostat microtome and Leica microdissection microscope (provided by the Pathobiology Lab, Jilin University, P. R. China); and 1,3-dimethyl propyl-3-ethyl carbodiimide (EDC) and N-hydroxyl succinimide (NHS) (Santa Cruz).

Purification of Annexin V-CdTe

EDC (500 ml) and NHS (500 ml) were added to CdTe nanocrystals (250 ml, 5 × 10^-4 mol/l density), and the mixture was agitated slowly for 30 min in the 37°C attemperator. The Annexin V antibody (1:50; 2500 ml, antibody titer 1:50) then was added to the mixed solution, which was slowly agitated for 3 h in the 37°C attemperator. The solution was added to a filter bag and refined at 4 ^o C for 12 h. The purified Annexin V-CdTe-conjugated polymer was stored at 4 ^o C. An ultraviolet spectrophotometer was used to measure the ultraviolet absorption spectrum of the Annexin V-CdTe-conjugated polymer and to obtain the ultraviolet absorption maximum band.

Sample preparation and Annexin V-CdTe staining

Adult male Wistar rats (n = 20, depuratory grade) weighing 250 ± 10 g were obtained from the Experimental Animal Center, The First Hospital, Jilin University, P. R. China. All animals were anesthetized and sacrificed by exsanguination. The experiments were performed with the approval of the Animal Experimentation Committee of Jilin University in accordance with the animal ethics guidelines published by the National Institutes of Health of China. The sciatic nerves were transected 2 cm above the transverse lines of the popliteal cavity, while the sural nerves were transected 2 cm above the line linked between the external and lateral malleolus. A small part of specimen of each nerve (20 mm) from the sciatic and sural nerves was prepared for immunohistochemical staining analysis (SABC) to verify the properties of peripheral nerve fascicles pre-experiment. The samples with a length of approximate 15 mm were then taken from each fascicle. Coronal sections (5 mm thickness) were made immediately, followed by immersion and staining in Annexin V-CdTe-conjugated polymer solution and incubation at 37°C in the attemperator. Coronal sections (5-mm thickness) of the sciatic and sural nerves were removed from the Annexin V-CdTe conjugated polymer 10, 15, 20, and 30 min after incubation and washed with PBS (0.01 mol/l) for 5 min. Optical qualities were detected under the fluorescence microscope at 10, 15, 20, and 30 min, respectively.

Results

The ultraviolet absorption maximum band of the Annexin V-CdTe-conjugated polymer was found at 587 nm, which is in the red fluorescence district [Figure - 1]. When using the fluorospectrophotometer to measure the fluorescence spectra, we found that the light emission spectrum was narrow and symmetric. When using 587 nm as the excitation wavelength, the CdTe's fluorescence peak position was located at 613 nm [Figure - 2].

To verify the sensory and motor fascicles in the peripheral nerves, we performed Annexin V-CdTe staining and observed the results under a light microscope. Based on the intraneural topography of the sural and sciatic nerves ^[3],[4] and the pre-experiment result of immunohistochemical staining analysis (SABC method), the sural nerves were positive for Annexin V-CdTe and appeared as red fluorescence when incubated for 10 min [Figure - 3]a, similarly, the sensory fascicles in the sciatic nerve exhibited a positive reaction as red fluorescence [Figure - 4]a, while in contrast, motor fascicles in the sciatic nerve showed no or little positive staining without red fluorescence [Figure - 4]a; however, at this time point the fascicles were not clearly stained [Figure - 3] and [Figure - 4]a. When the sections were incubated for 15 min, the staining was clearly visible. The sural nerves and sensory fascicles in the sciatic nerves exhibited a strongly positive reaction in the sections incubated for 15 min [Figure - 3]b and [Figure - 4]b, 20 min [Figure - 3]c and [Figure - 4]c, and 30 min [Figure - 3]d and [Figure - 4]d, whereas the motor fascicles showed no reaction [Figure - 4]b-d. No significant changes in the fluorescence intensity were observed among the samples incubated for 15, 20, or 30 min. We found that the red fluorescence was distributed mainly on the sensory fascicles [Figure - 3]b-d and [Figure - 4]b-d; in contrast, motor fascicles showed no or little positive staining [Figure - 4]b-d. Once did we see the red fluorescence during the operation, we can judge it as the sensory fascicles of the peripheral nerves, corresponding, we can judge it as the motor fascicles of the peripheral nerves by without red fluorescence. These results showed that we were able to identify the properties of peripheral nerve fascicles at 15 min point in vitro.

Discussion

The ability to differentiate between the sensory and motor fascicles is difficult due to the nature of the peripheral nerves. ^[14] Nevertheless, intraneural topography of various nerves at different levels and intraoperative sketches of the fascicular patterns, as demonstrated by Sunderland, ^[3] is helpful to surgeons. ^[4] However, cross sections of the proximal and distal stumps of a nerve, which suffer from a substantial traumatic defect due to sectioning, fail to show the location and morphology of sensory or motor fascicles. Moreover, nerve morphology alone is not helpful for their identification. To try to identify the fascicles, various electrophysiological methods have been applied during the course of an operation, including stimulation of the dissected fascicles of the distal and proximal stumps. ^[5],[6] However, electrophysiological methods require local anesthesia and are imprecise or unpleasant for the patients. Staining methods have been used extensively in previous studies. ^{[7],[8],[9],[10],[11],[12],[13]} However, differentiation of nerve fascicles based on measurements of carbonic anhydrase, acetylcholinesterase, choline acetylase, or choline acetyltransferase activity has limited applicability for several reasons. For example, incubation is time consuming, and Wallerian degeneration occurs after peripheral nerve injury, which results in a reduction of enzyme activities in the distal nerve stump. ^[35]

QDs are inorganic fluorophores that have the potential to overcome some of the functional limitations of organic dyes in biotechnological applications. They are substantially more photostable than conventional fluorophores, exhibiting high photostability, broad absorption, narrow and symmetric emission spectra, slow excited state decay rates, and large absorption cross sections. ^[36] These features enable concurrent imaging of multiple entities in a single biological experiment. ^[37] Compared with single organic fluorophores, QD probes do not suffer from intermittent on/off light blinking emission, and they are brighter and more stable against photobleaching. In addition, QDs often have a large stokes shift (i.e., a large separation between the excitation wavelength and the emission maxima), which can reduce autofluorescence and result in a several-fold increase in sensitivity compared to that of organic fluorophores. ^[38] Various sizes of QDs may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously. Nisman et al. used QDs in conjunction with immunogold to colocalize proteins at the ultrastructural level, ^[39] and tracking and imaging of nanocrystals in live animals have been achieved by Nie's group. ^[31] Kim et al. reported the use of near-infrared QDs to map lymph nodes during surgical procedures. ^[40] Although the feasibility of imaging with QDs has been demonstrated both in vitro and in vivo, ^{[25],[26],[29]} none of these studies have used a compatible polymer to differentiate between the sensory and motor fascicles.

Using covalently attached proteins, we demonstrated that CdTe QDs and Annexin V were biocompatible in vitro. These nanoconjugates were biocompatible and suitable for application in cell biology studies and immunoassays. In particular, CdTe QDs exhibit size-dependent tunable photoluminescence with narrow emission bandwidths (30-45 nm) that span the visible spectrum; they have also broad absorption spectra that allow simultaneous excitation of several particle sizes at a single wavelength. Preparation of the conjugate was simple, reproducible, and easily achieved. Moreover, we found that CdTe QDs bound at specific sites could be directly visualized by multicolor fluorescence microscopy. Over repeated scans, the CdTe-labeled samples showed very little photobleaching (far less than that seen with conventional dye molecules). Our results was consistent with Gotow's study; ^[41] however, inconsistent with that reported by Spreca, who showed that CdTe QDs labeling was significantly expressed in cellular membrane of Schwann cells and endochylema of the rat sciatic nerve. ^[17]

The improved photostability of Annexin V-CdTe-conjugated polymer will provide significant advantages in the real-time detection of Annexin V. Moreover, we succeeded in overcoming the main limitation of the slow biochemical reaction. To use this technique, we initially obtained nerve slices and marked the cut nerve ends for nerve repair 2 or 3 days later. Approximately 15 min of Annexin V-CdTe staining was then performed to precisely identify the sensory and motor fascicles based on fluorescent density. Finally, complete sensory and motor nerve repair was performed in one operation based on identified fascicles. Our finings showed that the 15-minute period required for staining was shorter than all other reported nerve identification procedures. ^{[7],[8],[9],[10],[11],[12],[13],[14]} Our result suggests that the Annexin V-CdTe-conjugated polymer immunostaining technique is an accurate and rapid method for distinguishing the sensory and motor nerve fascicles in the Peripheral nerves during microsurgery.

We have successfully established a method for the detection of Annexin V and for determining the properties of peripheral nerve fascicles. The CdTe-conjugated polymer is easily prepared and provides rapid detection, which makes it a good candidate for clinicians to further improve the results of interfascicular nerve anastomosis and grafting. This new procedure will enable surgeons to use Annexin V-CdTe-conjugated polymer to rapidly place nerve grafts at corresponding fascicles in the proximal and distal stumps. This procedure should be undertaken at a centre where microsurgical reconstruction is performed by highly skilled and experienced surgeons. In summary, Annexin V-CdTe-conjugated polymer is a useful tool for neurosurgeons to maximize the chances of successful nerve repair. This technology also will contribute to doing research on tracing peripheral nerves and the plasticity of peripheral nerves after nerve injury.

References

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