| Part 1. The clinical research about HBO on the growth of rat intracranial glioma modelsPurpose:To study the effect of HBO on the growth of rat intracranial glioma models.Methods:Totally 40 male SD rats(weight 250～280g) were injected in the right caudate nucleus with C6 cell suspension(10ul), the concentration of which is 105/ul. The HBO treated animals (20 rats) received a therapy of 3.0 ATA with 100%02 and maintained for 1h, respectively on 2d,4d,6d,8d, 10d,12d after the injection; while the control animals (20 rats) were just put into the HBO chamber but received normbaric room air for the same time as the HBO treated rats. All treated and control animals went through neurological function tests of bilateral forelimbs through the "vibrissae-stimulated forelimb placing test" on 1d,3d,5d,7d,9d, 11d,13d after the injection. On 16d post the injection, all rats weighed to calculate weight change,5 rats of each group were selected at random and received head MRI to evaluate the tumor volume. Then 10 of each group were over-anesthetized, and their brains were removed to determinate the tumor weight and brain water content, the rest rats waited for the natural death time so that we could calculate the average survival days, the shortest and the longest survival in each group.Results:1. After establishing SD-C6 intracranial glioma model of rats through the stereotactic caudate injection, there was a significant decrease of the left forelimb function score in both treated and control animals on Id post the injection, and there was no difference between the two groups(P>0.05). After the HBO therapy just for 1 time, however, the left limb neurological function score in treated animals is obviously higher than that in control animals on 3d post the operation(P<0.01), and the difference persisted to 13d post the operation. The rats in both groups began to behave a decrease of the right forelimb function score on about 7d post the operation, and the score in treated animals is higher than that in control animals from 9d to 13d after the operation(P<0.01).2. The ponderal growth of treated animals on 16d post operation was 11.0±10.5g, while 18.2±9.8g in control animals, and there was no significant difference between the two groups(P>0.05).3. On 16d post the operation, the head MRI scan showed the tumour occupying in both treated and control animals on 16d post the operation, but the tumor volume of treated animals was 0.410±0.121cm3,larger than that of control animals (0.272±0.135cm3) (P<0.05).4. On 16d post the operation, the tumor weight of treated animals was 0.26g±0.08g, higher than that of control animals--0.20g±0.11g (P<0.05).5. On 16d post the operation, the water content of the right hemisphere was significantly higher than that of the left hemisphere in both groups, but the water content of bilateral hemisphere and cerebellar between two groups had no significant difference(P>0.05).6. The 4 weeks survival rate of HBO treated animals is 50%(5/10), and the shortest and the longest survival were 23d and 35d; the 4 weeks survival rate of control animals is 60%(6/10), and the shortest and the longest survival were 24d and 37d. There was no obvious difference in survival between the two groups(P>0.05).Conclusions:1. HBO (3ATA, 1h×6 times) may play a role in promoting tumor growth on the C6 rat intracranial glioma model.2. There is a significant rehabilitation efficacy of HBO on neurological deficits caused by intracerebral tumor growth and operative injury.3. HBO (3ATA, 1h×6 times) has no significant effect on survival of the C6-SD rats. Part 2. HBO on VEGF/HIF-1αexpression, angiogenesis and apoptosis of tumor cells in rat intracranial glioma modelsPurpose:To study the effect of hyperbaric oxygen on the expression of VEGF/HIF-1α, angiogenesis and tumor cell apoptosis in rat intracranial glioma models.Methods:Totally 20 male SD rats(weight 250-280g) were injected in the right caudate nucleus with C6 cell suspension(10ul), the concentration of which is 105/ul. The HBO treated animals (10 rats) received a therapy of 3.0 ATA with 100% O2 and maintained for 1h respectively on 2d,4d,6d,8d, 10d,12d after the injection; while the control animals (10 rats) were just put into the HBO chamber but received normbaric room air for the same time as the HBO treated rats. All the rats were sacrificed and removed their brains after over-anesthesia and cardiac perfusion.Then the fixed tissue successively passed through paraffin imbedding, brain slices, HE staining, immunohistochemical staining to determine the expression of vWF/VEGF/HIF-1αintratumor and surrounding the tumor, and the expression of vWF standed for the microvessel density. Then TUNEL stained apoptotic cells of intratumoral and peritumoral regions were acessed.Results:1. HE staining confirmed tumor specimen in all tested tissues. The tumor cells were spindle-shaped into density, with large and deep stained nuclear, and the typical nodules were visible, which arranged into whirlpool nest in the tumor. Some tumor cells infiltrated the surrounding normal tissues but the boundaries were still clear, and some tumors had formed a outer membrane. However, there was a more intensive arrangement of tumor cells, a more vigorous proliferation and a more common angiogenesis in the tumors of HBO treated animals, while a more widespread necrosis and hemorrhage in tumors of control group.2. The intratumoral expression of VEGF was significantly higher in HBO group(P<0.05), and there was rare expression around the tumor and in normal tissues. Meanwhile, the intratumoral expression of HIF-1αwas also significantly higher in HBO group(P<0.05), and no significant difference in peritumoral expression between two groups(P>0.05).3.The intratumoral microvessel density was significantly higher in HBO group(P<0.05), and the peritumoral microvessel density of no significant difference between two groups(P>0.05).4. TUNEL label fluorescence imaging showed the number of positive cells intral and around the tumor tissues of HBO group was significantly lower than that of the control group. DAB-stained confirmed intratumoral and peritumoral apoptotic cell ratio of control group were significantly higher than that of HBO group(P<0.05).Conclusions:HBO could up-regulate the expression of vWF, HIF-1a, VEGF in rat brain glioma models, and inhibit apoptosis of glioma cells and peritumoral cells.
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