Correlation between retinal ganglion cell loss and nerve crush force-impulse established with instrumented tweezers in mice.

Objectives: Rodent models of optic nerve crush (ONC) have often been used to study degeneration and regeneration of retinal ganglion cells (RGCs) and their axons as well as the underlying molecular mechanisms. However, ONC results from different laboratories exhibit a range of RGC injury with varying degree of axonal damage. We developed instrumented tweezers to measure optic nerve (ON) crush forces in real time and studied the correlation between RGC axon loss and force-impulse, the product of force and duration, applied through the instrumented tweezers in mice.Methods: A pair of standard self-closing #N7 tweezers were instrumented with miniature foil strain gauges at optimal locations on both tweezers' arms. The instrumented tweezers were capable of recording the tip closure forces in the form of voltages, which were calibrated through load cells to corresponding tip closure forces over the operating range. Using the instrumented tweezers, the ONs of multiple mice were crushed with varied forces and durations and the axons in the immunostained sections of the crushed ONs were counted.Results: We found that the surviving axon density correlated with crush force, with longer duration and stronger crush forces producing consistently more axon damage.Discussion: The instrumented tweezers enable a simple technique for measurement of ONC forces in real-time for the first time. Using the instrumented tweezers, experimenters can quantify crush forces during ONC to produce consistent and predictable post-crush cell death. This should permit future studies a way to produce nerve damage more consistently than is available now.

Standardizing optic nerve crushes with an aneurysm clip.

OBJECTIVES: Despite the widespread use of optic nerve injury models to simulate central nervous system injury, model protocols vary from laboratory to laboratory, making it difficult to directly compare findings between studies. METHODS: To standardize the optic nerve crush injury model, the commercially available Yasargil aneurysm clip, which provides a consistent clamping force, was used to produce a crush injury to the rat optic nerve. Histology was verified with hematoxylin-eosin. The number of retinal ganglion cells (RGCs) was counted by fluorescent gold dye labeling. RESULTS: Following nerve crush injury, the density of RGCs was substantially reduced in the aneurysm clip-operated group relative to the normal and sham-operated groups, and no discernable difference was noted between the latter two control groups. DISCUSSION: The present findings suggest that Yasargil aneurysm clip effectively produces permanent injury to the optic nerve with evidence from retrograde tracing of RGCs and may provide a standard technique for optic nerve crush studies.

Morphologic and morphometric evaluation of experimental acute crush injuries of the sciatic nerve of rats.

In order to qualify and quantify nerve fiber lesion following an acute crush injury, a morphologic and morphometric study was carried out in 25 Wistar rats divided into five groups of five animals each according to the crushing load applied, i.e., 500, 1,000, 5,000, 10,000, and 15,000 g. The injury was produced under general anesthesia on a 5mm-long intermediate segment of the right sciatic nerve for 10 min using a dead-weight machine. The animals were killed with an excessive dose of anesthetics 72 h later and submitted to perfusion with a fixing solution through the abdominal aorta immediately after death. Both the right and left sciatic nerves were removed and prepared for histologic and morphometric examinations; 5 microm-thick sections stained with 1% Toluidine blue were examined under a light microscope equipped with a video camera linked to a computer loaded with a graphic program (KS 400). The morphometric studies included measuring total number of fibers, fiber density, fiber diameter, myelin fiber area, axon diameter, axon area and G ratio. The results showed that damage to the nerve fibers began to appear as early as with the 500 g load and was similar in all groups despite the load applied, increasing with the 10,000 and 15,000 g loads, although the external supporting tissues and small diameter fibers were preserved. The predominant type of lesion produced was axonotmesis.

A mild acute compression induces neurapraxia in rat sciatic nerve.

The pressure that induces neurapraxia in rat remains unrevealed. To determine the appropriate force to induce neurapraxia, two types of clips were applied to the sciatic nerve and were evaluated with functional, electrophysiological, and histological examinations. With a compression of 60 g/mm2, walking track analysis showed complete sciatic nerve paralysis one day postoperatively, but became normal in 14 days. Electrophysiologically, complete conduction block occurred one day post operatively, whereas the motor conduction velocity (MCV) below the compression site remained normal. Histologically, only limited signs of Wallerian degeneration were seen. The model in this study exhibited the features of neurapraxia.

A temporal study of axonal degeneration and glial scar formation following a standardized crush injury of the optic nerve in the adult rat.

PURPOSE: In this methodological study, we describe a standardized optic nerve injury model in adult rats. The temporal pattern of the glial response is mapped in relation to axonal regeneration and degeneration. METHODS: A standardized optic nerve crush injury was made in adult rats, using a fine pair of forceps with a preset pressure of 0.6 N during 10 seconds. RESULTS: This crush injury resulted in a pronounced astroglial and microglial proliferation 2-7 dpi. Degenerative axons distal to the lesion presented a gradual decrease in neurofilament immunoreactivity. In contrast, Fluoro-Jade staining was gradually increased in these degenerative axons from 2-7 dpi. Regeneration was observed proximal to the lesion in sprouts double labeled with the tracer Fluoro-Gold and anti-GAP43. CONCLUSION: The standardized optic nerve crush injury model used in the present study resulted in a reproducible degenerative pattern including a pronounced astroglial and microglial proliferation. The gradual decrease in Neurofilament immunoreactivity in degenerative axons, and a gradual increase in Fluoro-Jade staining was paralleled with a spontaneous regeneration proximal to the lesion, shown with tracing and GAP43-immunoreactivity. The combination of these markers and this experimental setup thus allows analysis of the entire degenerative/regenerative process in a standardized CNS axotomy model.

Yasargil-Phynox aneurysm clip: a simple and reliable device for making a peripheral nerve injury.

A great number of devices were used to make a peripheral nerve injury. In the scientific literature, experimental crush injuries have been usually created using forceps or hemostatic forceps, neither of which allows quantitative or standard application of compression. Therefore, we used a Yasargil-Phynox aneurysm clip to make a reliable and standardized peripheral nerve injury. The advantages and disadvantages of this clip were discussed. In particular, we think that standardization of the compression is necessary to compare interlaboratory results.

Standardizing nerve crushes with a non-serrated clamp.

Standardized experimental nerve crush attempts should include the number, duration, and intensity (amount of pressure) of crushes. The authors have developed a new crushing device, a clamp with which predetermined forces can be applied to nerves. This allows the exertion of different, standardized forces to crush a nerve within a scale that produces second-degree injuries. The main advantages of the clamp are that it is small, although very robust, is purely mechanical, and is easy to handle. The jaws of the clamp are not serrated, so that pressure on the nerve is uniformly transmitted. To avoid unintended nerve damage, the edges of the jaws are smoothly rounded off. The closure of the clamp is mechanized by a spring. As the spring is exchangeable, any number of different preloads are available. The force can be varied, according to different requirements, and is applicable to variantly thick nerves in any experimental animal, thus enhancing standardization, and making cross-over comparisons of experimental study results possible.