The 9th and 10th spinal nerves of adult (60 ± 12 g) anesthetized grass frogs (R. pipiens) of both sexes were unilaterally transected about 5 mm distal to their exit from the spinal column. In those experiments involving stump removal, the severed nerves distal to the site of transection were dissected from the retroperitoneal space and thigh on the same side and removed. Seven days after axotomy, tissues were fixed by intracardiac perfusion and overnight immersion in cold 4% p-formaldehyde, and the control and experimental lumbar spinal cords were cryosectioned at 10µm and every fifth section was stained with cresylecht violet, and subjected to morphometric analysis. In each section examined, areas were defined for all motoneurons with a visible nucleolus, using a Technology Resources Imagemaster 1000 system calibrated with 9µm diameter plastic beads (Flow Cytometry Standards, Inc.). In the frog, lumbar motoneurons are easily distinguished by their size from other neurons in the ventral horn. Gamma motoneurons are not found in the frog spinal cord (7). The perimeter of each nucleolus, nucleus and cell body was traced three times with a mouse-driven cursor and the mean of the three computed areas was converted to µm².

I.The Swelling Response in Lumbar Motoneurons to Axotomy is Strictly Unilateral.

In the presence of the distal stump, the swelling response of motoneuronal nucleoli, nuclei and cell bodies is observed only on the operated side of the lumbar spinal cord. (Table 1). No significant size differences for these three structures were observed between contralateral and unoperated motoneurons.

Table 1. Nucleolar, nuclear and cell body areas (mean ± S.D.) in lumbar motoneurons 7 days after unilateral spinal nerve transection. Data from 7 operated frogs (331 motoneurons analyzed) and 6 unoperated frogs (446 motoneurons analyzed). a = p < 0.01 vs. contralateral values by Student's paired t-test (df = 6). b = p < 0.01 vs. unoperated controls by Student's unpaired t-test (df = 11).

                      Area(µm2), Distal Stump Left In
          Ipsilateral Contralateral Ipsi/Contra  Unoperated  Ipsi/Unop
    
Nucleolus   25.3± 3.4     15.9± 2.3     159.1%    17.6± 0.9     143.8%
              a,b
Nucleus    195.7± 36.3   183.6± 20.6    106.6%   173.3± 8.6     112.9%
               a
Cell body 1360.2± 192.8 1014.2± 94.7    134.1%  1040.5± 103.9   130.7%  
              a,b

II. The Magnitude and Unilaterality of Swelling are Not Influenced by Removal of the Distal Stump.

Comparison of the data in Table 1 and Table 2 (below)shows that removal of the distal stump affects neither the magnitude of the mean swelling responses of nucleoli, nuclei or cell bodies on the ipsilateral side of the spinal cord nor the absence of a swelling response in contralateral motoneurons.

Table 2. Mean areas (± S.D.) in motoneurons 7 days following removal of distal stump (635 motoneurons from 15 frogs). a = p< 0.01 vs. contralateral values by Student's paired t-test (df = 6). b = p < 0.01 vs. unoperated controls by Student's unpaired t-test (df = 11).

                     Area(µm2), Distal Stump Removed
               Ipsilateral       Contralateral       Ipsi/Contra

Nucleolus        25.8± 2.1         16.9± 1.3             152.7%

Nucleus         215.9± 19.4       183.9± 17.7            117.4%

Cell Body      1453.9± 85.6      1098.6± 79.9            132.3%

III. Lumbar Motoneurons of All Sizes Swell Proportionately After Axotomy.

The Role of the Distal Stump in Perikaryal Responses to Axotomy. The distal stump influences changes in axotomized motoneurons in a selective and still incompletely understood way. Wu et al. (8) found evidence that the distal stump prevents nitric oxide synthase induction in cervical motoneurons and promotes their survival after axotomy. Our data indicate that the distal stump does not initiate the swelling responses in injured lumbar motoneuron cell bodies. Moreover, a study by Ochs et al.(9) found that the distal stump does not initiate chromatolysis within the injured motoneuron cell bodies. Whether the distal stump is a source for other long-distance responses to axotomy, such as the increases in circulating cytokines and transcriptional responses in spared motoneurons, remains to be shown.

The Mechanism and Functional Significance of Swelling Responses in Axotomized Lumbar Motoneurons. Despite a century of study, most of the classic morphological responses of the neuronal cell body and dendrites to axonal injury have not been explained. Depending upon the specific neuron, those responses can include perikaryal, nuclear and nucleolar swelling or atrophy, chromatolysis, nuclear eccentricity and dendritic atrophy (10). With regard to the swelling phenomena described here, it is not known how they are produced and whether they support or interfere with regeneration of the injured motor axon. Our study indicates that, once identified, the mechanism for swelling under the present conditions will be refractory to removal of the distal stump. Furthermore, it will exist only ipsilaterally, will operate uniformly upon injured motoneurons of all sizes, and will produce coordinated changes in nucleolar, nuclear and perikaryal size consistent with the magnitude and time course of swelling observed here. Once understood, the swelling mechanism may then shed light on the relationship of this particular injury response to axon regeneration.

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