It is hypothesized that group I animals will show equal amounts of labeled neurons in both the left and right red nuclei. For group II animals it is hypothesized that no labeling will be seen in the left and right RN. For group III animals it is hypothesized that the both the left and right red nuclei will show a small number of FG labeled neurons, indicative of regeneration. Finally, it is hypothesized that group IV animals will show a large mass labeled FG neurons in both red nuclei, strongly indicating regeneration.

MATERIALS AND METHODS

Adult male Wistar rats were used for this study. The rats were removed from their cages and anesthesia was induced with a 5% isoflurane/oxygen mixture and maintained at a 2.5% isoflurane/oxygen level. The rats were then shaved at the marked site of incision and betadine solution was applied to prevent microbial infection. The rats were placed, belly-side down, under a surgical microscope. A No. 10 scalpel blade was used to separate the neck musculature along the midline and expose the vertebral column at the cervical level (C4/C5). After the protective dura layer was opened, and the spinal cord revealed, a right rubrospinal tract lesion was made with a No. 11 scalpel blade. The implant was placed into the lesion cavity using small forceps. The wound area was closed using surgical staples and the animals were returned to their cages.

Six weeks after the first surgery, the rats were re-anesthetized and the neck musculature was again separated along the midline using a No. 10 scalpel blade. For this second surgery, the vertebral column and spinal cord were exposed at C6/C7, two levels below the lesion site, using a No. 11 scalpel blade. Approximately 1 _l of the retrograde tracer, fluorogold (FG), was directly injected into the right rubrospinal tract using a Hamilton syringe. Following injection, the wound area was closed with surgical staples and the rats were placed into their respective cages. Figure 1 illustrates the pathway of the rubrospinal tract (RST) as well as the lesion site and FG injection site.

Figure 1. Rat rubrospinal tract. This figure shows the contralateral and ipsilateral projections from the red nuclei, as well as the lesion site at cervical spinal cord level C4/C5 and FG injection site at level C6/C7.

Three days later the rats were sacrificed with an overdose of sodium pentobarbital (100 mg/kg). The entire brain and spinal cord were carefully removed and placed into vials containing 0.1M phosphate buffer saline. The brain was sectioned at 50 μm, mounted onto gelatin-coated glass, and left to dry overnight. Then the slides were examined using a fluorescent microscope, where the FG labeled RN neurons could be analyzed.

RESULTS

Group I animals showed one pattern of labeling in the red nuclei. In these experiments animals 1 and 2 showed a large mass of FG labeled neurons in both the left and right red nuclei (Figure 2).

Figure 2. Schematic representation of FG labeled red nucleus neurons of group I animals (control). Note the equal numbers of FG labeled neurons (red dots) in both nuclei.

Group II animals showed two patterns of FG labeling in the red nuclei. Animal 1 showed very few FG labeled neurons in the left RN and a large mass of labeled neurons in the right RN (Figure 3A). The second pattern of labeling of group II animals was seen in animals 2,3,4, and 5. All of these animals showed results very similar to the control animal with a relatively equal, large quantity of labeled neurons in both the left and right red nuclei (Figure 3B).

Figure 3. Schematic representation of FG labeled red nucleus neurons (red dots) of group II animals. A. Note the decreased number of FG filled neurons in the left (lesioned) nuclei. B. Animals shows a similar labeling pattern to group I animals (controls).

In group III animals, two different FG labeling patterns were seen in the red nuclei. Animals 1 and 3 showed larger quantity of neurons labeled in the left RN as compared to the right RN (Figure 4A). The second FG labeling pattern seen in group III involves only animal 2. Both red nuclei were labeled in this animal, however the left RN had a smaller quantity of FG labeled neurons as compared to the right RN (Figure 4B). Animals 4 and 5 did not show any FG labeling.

Figure 4. Schematic representation of FG labeled red nucleus neurons (red dots) of group III animals. A. Note the decreased number of FG filled neurons in the right RN as compared to the left (lesioned) RN. B. The animals show a larger number of FG labeled neurons in the right RN as compared to the left (lesioned) RN.

Group IV animals showed one major pattern of labeling. All animals showed a large, equal mass of neurons labeled with FG in both the left and right RN.

Figure 5. Schematic representation of FG labeled red nucleus neurons (red dots) of group IV animals. Animals show a similar labeling pattern to group I animals (controls).

DISCUSSION

Group I animals (the control group) receiving only an injection of the FG tracer showed one pattern of labeling in the red nuclei. In these experiments, animals 1 and 2 showed a large number of FG labeled neurons in both the left and right red nuclei. The quantity of labeled neurons in each RN were approximately equal in number. This labeling was due to retrograde transport of FG up the uninjured right RST or diffusion of the FG tracer across the spinal cord into the left RST. This pattern of labeling was consistent with the hypothesis for group I animals.

Group II animals, which received only the right rubrospinal tract (RST) lesion, showed two patterns of FG labeling in the red nuclei. Animal 1 showed very few FG labeled neurons in the left RN and a large mass of labeled neurons in the right RN. This labeling was most likely seen due to leakage and diffusion of the FG tracer into the left RST or the lesion site. The very large difference in the number of neurons labeled in the red nuclei may indicate a somatotopic arrangement of axons in the RST. This first pattern of labeling may have also resulted from FG diffusion into the lesion site of the right RST.

The second pattern of RN labeling of group II animals was seen in animals 2,3,4, and 5. All of these animals showed results very similar to the control animal with a relatively equal, large quantity of labeled neurons in both the left and right red nuclei. This labeling was most likely due to leakage and diffusion of the FG tracer into the left RST or the lesion site. This second pattern of labeling may have also resulted from FG diffusion into the lesion site, causing the severed axons to take up FG to their respective red nuclei. These results did not match the hypothesis for group II animals.

In group III animals, which received a lesion followed by the alginate implant, there were two different FG labeling patterns seen in the red nuclei. Animals 1 and 3 showed a larger number of neurons labeled in the left RN as compared to the right RN. This labeling may be due to diffusion of FG across the spinal cord into the left RST, diffusion of FG into the lesion site, or regeneration of left RN neurons. Somatotopic arrangement of rubrospinal axons may have played a role in the larger number of FG labeled neurons in the left RN versus the right RN.

The second FG labeling pattern seen in group III involves only animal 2. Both red nuclei were labeled in this animal, however the left RN had a smaller quantity of FG labeled neurons as compared to the right RN. This result also may be due to FG diffusion or regeneration. This labeling pattern may also be due to FG leakage into the lesion site in the right RST, thus causing the red nuclei to be labeled. Surprisingly, animals 4 and 5 did not show any FG labeling at all. This may be due to an improper injection of the FG into an area other than the RST or spinal cord. These results did not completely match the hypothesis for group III animals. However, it cannot be definitively concluded that the FG labeled neurons seen in the red nuclei were due to regeneration.

Group IV animals, which received a lesion followed by the alginate implant with microglia showed one major pattern of labeling: all animals showed large numbers of neurons labeled with FG in both the left and right RN. These results looked very similar to the control animals. This labeling pattern may be due to either diffusion or regeneration. These results matched the hypothesis for group IV animals. Additional work using a more refined tract tracing technique will be required to confirm that these results are due to successful regeneration.

In summary, the present study investigated the use of biomaterials to induce regeneration in the central nervous system. It was discovered that the volume and method of delivery of FG used in these experiments did not allow unequivocal conclusions regarding regeneration of CNS axons. Several past studies have used FG in their studies concerning CNS regeneration. Kobayashi et al. and Liu et al. used 1.0 μl FG in their regeneration studies with the rubrospinal tract [2, 3]. The results from the present study also used 1 μl FG tracer and therefore casts doubt on the regenerative findings by other researchers using the same quantity of FG. In future studies, the volume and delivery of FG injected into the rubrospinal tract will need to be modified in order to successfully eliminate the problem of diffusion.

REFERENCES

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