Effects of pyruvate on cell morphology and Nissl bodies in L5 DRG tissues
HE staining
In the PYR group, the DRG nervous cells showed only mild swelling and a small amount of cytoplasm vacuoles. In contrast, in the SUS and SAL groups, the DRG cells presented quite swelling, gap widened in varying degrees and some cytoplasmic vacuoles and numerous fine granular basophilic materials among cells.
These changes appeared more significant in the SUS and SAL groups than in the CON and PYR groups. The cell morphological appearance in Group PYR was slightly changed in comparison with that in Group CON (Fig. 1).
In the PYR group, the DRG neurons showed only mild swelling and a small amount of cytoplasm vacuoles. In the SUS and SAL groups, the neurons presented quite swelling, gap widened in varying degrees and some cytoplasmic vacuoles and there were numerous fine granular basophilic materials among the cells. The cell morphological appearance in Group PYR was slightly changed in comparison with that in Group CON (scale bars 25μm).
Nissl body in neuron
In the CON and PYR groups, Nissl basophilic granules were abundant with near normal appearances in neurons. However, in the SUS and SAL groups, the morphological size and the distribution pattern of Nissl bodies were smaller and scattered, which were lightly stained. In some DRG cells, the Nissl bodies also appeared disassociated. In addition, the observed and quantified changes were more significant in the SUS and SAL groups than in the CON and PYR groups and there was no significant difference in numbers of Nissl body stained neurons between Groups CON and PYR (Fig. 2 A and B).
In the CON and PYR groups, Nissl basophilic granules were abundant with near normal appearance. However, in the SUS and SAL groups, the Nissl bodies were lightly stained with smaller morphological sizes and scattered distribution. In some DRG neurons, the Nissl bodies also appeared disassociated in Groups SUS and SAL (scale bars 100μm).
The amounts of Nissl-stained neurons/1.0 mm2 in five random fields were quantified by using a light microscope (X400; five section slices were evaluated in average in each rat; n = 10). In addition, the observed and quantified changes were more obvious in the SUS and SAL groups than in the CON and PYR groups and there was no significant difference in numbers of Nissl body staining neurons between Groups CON and PYR. There were statistically significant differences between Groups SUS and SAL from Group PYR, respectively (**P<0.01).
Degenerated-MBP expression
In the SUS and SAL groups, the cell number of dgen-MBP was increased; the cells were swollen and their boundaries were unclear with heavy fluorescent in the immunofluorescence detection. These changes did not obviously appear in the CON and PYR groups (Fig. 3 A). The mean OD value of dgen-MBP was significantly increased in Groups SUS and SAL, compared with Groups CON and PYR. As shown in Fig. 3 B, there were statistically significant differences between Groups SUS and SAL from Groups CON and PYR (P<0.01), however, there was no difference between both later groups (P>0.05) in the mean OD value.
The SUS and SAL groups showed more expressions of dgen-MBP than the CON and PYR groups (scale bars 50μm).
Mean OD values of dgen-MBP immunofluorescence staining in L5 DRG were higher in Groups SUS and SAL than in Groups CON and PYR. There were statistically significant differences between Groups SUS and SAL from Group PYR, respectively (**P<0.01).
dgen-MBP: degenerated myelin basic protein
Spinal cord scan
In the CON groups, the gray matter and white matter areas in L5 were normal. In the SUS and SAL groups, the areas of gray matter and white matter were atrophy. These alterations appeared more significant in the SUS and SAL groups than in the PYR group that was close to Group CON. The relative area ratios of gray matter in the HU groups to the CON group also illustrated a significant decrease in Groups SUS and SAL, but a comparable outcome in Groups PYR and CON (Fig. 4 A and B).
In Group CON and PYR, the posterior part of the gray matter dorsal horn was slightly thicker and the outer layer was gentle; the edge of the anterior horn of the gray matter was enlarged and smooth. In Group SUS and SAL, the middle and lateral parts of the gray matter dorsal horn were slightly curved and the out layer was steep. In Group SUS, the medial margin of the anterior horn was thinner; in Group SAL, the anterior horn of the gray matter was significantly reduced (scale bars 1μm).
The gray matter area was bigger in the PYR group than in the SUS and SAL groups. The alterations appeared more significant in the SUS and SAL groups than in the CON and PYR groups and they were close to each other in Groups CON and PYR. The gray matter areas were statistically significant differences between SUS and SAL from PYR groups, respectively (**P<0.01).
Effects of pyruvate on GDNF and GFAP expressions in L5 DRG tissues
GDNF protein
As shown in Fig. 5, 8 weeks of simulated microgravity induced a significant reduction in GDNF expressions. Compared with the CON group, the relative GDNF protein content in the DRG was reduced by 21% and 20% in the SUS and SAL groups, respectively. In contrast, the GDNF content was 123% and 112% higher in the PYR group than in the SUS and SAL groups, respectively. There were statistically significant differences between SUA and SAL groups from CON and PYR groups (P<0.01). However, the protein content was significantly higher in group PYR than in group CON (p<0.01).
Simulated microgravity induced significant reduction in GDNF expressions. There were statistically significant differences between SUA and SAL groups from CON and PYR groups. However, the protein content was significantly higher in Group PYR than in Group CON. There were statistically significant differences between Groups CON, SUS and SAL from Group PYR, respectively (**P<0.01).
GFAP expression
The relative GFAP protein expression was significantly increased in all three suspension groups, compared with Group CON, as shown in Fig. 6. GFAP contents were 2.5 times and 2.3 times in Groups SUS and SAL over Group CON, respectively. However, the GFAP protein in Group PYR was reduced by 28% and 24%, compared to Groups SUS and SAL, respectively. There were statistically significant differences between Groups SUA and SAL from Groups CON and PYR (P<0.01). And the GFAP level was still significantly higher in Group PYR than in Group CON (P< 0.01).
The GFAP protein expressions were significantly increased in all three suspension groups. There were statistically significant differences between Groups SUA and SAL from Groups CON and PYR. And the GFAP level was still significantly higher in Group PYR than in Group CON. There were statistically significant differences between CON, SUS and SAL from Group PYR, respectively (**P<0.01).
Effects of pyruvate on ATP and ATPase expressions in L5 DRG tissues
ATP content
The adenosine triphosphate (ATP) content was decreased in all three suspension groups, compared to Group CON. ATP contents (nmol/gprot) were reduced by 60% and 61% in Groups SUS and SAL from Group CON, respectively. However, they were 35% and 38% higher in the PYR group than in the SUS and SAL groups, respectively. There were statistically significant differences between Groups SUS and SAL from Groups CON and PYR (P<0.05; P<0.01; Fig. 7 A). However, the ATP level was still significantly lower in Group PYR, compared with Group CON (P<0.01).
The ATP contents were decreased in all three suspension groups. There were statistically significant differences between Groups SUA and SAL from Groups CON and PYR. However, the ATP level was still significantly lower in Group PYR compared with Group CON. There was a statistically significant difference between Group SUS and Group PYR (P*<0.05) and significant different between Groups CON and SAL from Group PYR, respectively (**P<0.01).
ATPase activity
The adenosine triphosphatase (ATPase) activity was reduced in all three suspension groups relative to Group CON. ATPase activities (μmol/mgprot/h) in Groups SUS and SAL were decreased by 57% and 59% from Group CON, respectively. However, they were 42% and 51% higher in the PYR group than in the SUS and SAL groups, respectively. There were statistically significant differences between the SUS and SAL groups from the CON and PYR groups (P<0.01; Fig. 7 B). But, the ATPase activity in Group PYR was still significantly reduced relative to Group CON (P<0.01).
The ATPase activity was reduced in all three suspension groups. There were statistically significant differences between the SUS and SAL groups from the CON and PYR groups. But, the ATPase activity in Group PYR was still significantly reduced relative to Group CON. There were statistically significant differences between Groups CON, SUS and SAL from Group PYR, respectively (**P<0.01).
The rats’ diet, defecation and general activity in the experiment were normal, no tail injury and death occurred.