KE Yi-quan(柯以铨), XU Ru-xiang(徐如祥), CHEN Chang-cai(陈长才). Department of Neurosurgery, Zhujiang Hospital, First Military Medical University, Guangzhou 510282 中华创伤杂志 1998 2 14 1
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Objective To assess the effects of level changes of 5-hydroxytryptamine (5-HT) and dopamine (DA) in cerebral microvasculature on occurrence of secondary damage in traumatic brain injury in rats.
Methods The level changes of 5-HT and DA in the cerebral microvasculature were determined with high performance liquid chromatography using electrochemical detector (HPLC-ECD).
Results The levels of 5-HT and DA in the cerebral microvasculature were significantly elevated 30 minutes after brain injury (P?.01) and gradually decreased and were lower than the control level 48 to 72 hours postinjury. The water content of brain tissue was significantly increased 30 minutes postinjury and came down to the control level 168 hours after injury.
Conclusion The significant increase of 5-HT and DA levels in the cerebral microvasculature is one of the important factors to induce secondary damage after brain injury because it can result in the functional disturbance of microvascular rela-
xation and impairment of blood-brain-barrier.
In recent years, it has been reported that the metabolic disorder of monoaminergic neurotransmitters is an important factor to induce secondary damage after brain injury. In order to investigate whether the changes of 5-hydroxytryptamine (5-HT) and dopamine (DA) play a role to induce secondary brain damage after brain injury, the levels of the 2 agents were determined in rats with high performance liquid chromatography using electrochemical etector (HPLC-ECD) after brain traumatic injury.
MATERIALS AND METHODS
Two hundred and twenty adult male Sprage-Dawley rats, weighing 250±30 g, were randomized into 3 groups as follows: the normal control group of 20 rats without any intervention, the sham-operated group of 20 rats undergoing a surgical procedure without brain injury, who were killed 24 hours after surgery, and the brain-injured group of 180 rats sustaining brain injury. Twenty rats were killed 30 minutes, 1, 2, 3, 6, 24, 48, 72 and 168 hours respectively after injury. Half of the rats were used for the determination of 5-HT and DA levels in the cerebral microvessels and the other half for that of water content of the brain tissue.
The rat model of brain injury was established with Feeney's method1 with modification. Local contusion was inflicted over the left parietal region of 12.6 mm2 and the strength of the impacting force was 0.0075 N (750 g·cm).
The cerebral microvessels were isolated with the method reported by Goldstein et al2 and it was slightly modified according to our experience. The protein content of cerebral microvessels was determined with Lowry's method.
The levels of 5-HT and DA were measured with the HPLC-ECD system manufactured by the Shimdzu Corporation of Japan and Kim's method3 . The mobile phase comprised 80 mmol/L of disodium hydrogen orthophosphate, 60 mmol/L of citric of acid buffer, 0.21 mmol/L of sodium octane sulfonic acid,
0.22mmol/L of EDTA and 20% (v/v) of methanol at pH 4.3. The flow rate was maintained at 1.5 ml/min. The levels of 5-HT and DA were expressed in ng/mg of protein of the cerebral microvessels.
The water content of the brain was determined after the rats were killed. A piece of the grey matter and a piece of the white matter weighing (100±10) mg were cut from the posterior edge of the contused region. After the wet weight was measured with an electron balance (accuracy of 0.1mg), the tissue samples were desiccated in a 110° desiccator for 24 hours and the dry weight was measured. The percentage of water in the grey and white matter was calculated respectively and the remained brain tissue was routinely prepared for pathological study.
All the data were statistically processed with Student's t-test and were expressed in ±SD.
RESULTS
The levels of 5-HT and DA in the cerebral microvasculature significantly increased from 30 minutes to the 6th hour postinjury and then came down gradually and became lower than the control level in the 48th and 72nd hours postinjury (Table 1).
Table 1 5-HT and DA levels in cerebral microvasculature after
brain injury (ng/mg of microvascular protein)
| Groups | n | 5-HT | DA |
| Normal control | 10 | 35.83±3.53 | 71.39±8.03 |
| Sham-operated | 10 | 35.25±5.38 | 71.77±7.14 |
| Brain-injured | | | |
| 0.5 h postinjury | 10 | 134.84±22.05# ▲ | 202.62±34.66# ▲ |
| 1 h postinjury | 10 | 56.57±4.32# ▲ | 708.61±69.44# ▲ |
| 2 h postinjury | 10 | 73.34±4.77# ▲ | 457.08±45.58# ▲ |
| 3 h postinjury | 10 | 73.17±4.22# ▲ | 481.99±40.87# ▲ |
| 6 h postinjury | 10 | 64.82±4.36# ▲ | 416.14±29.33# ▲ |
| 24 h postinjury | 10 | 60.29±5.31# ▲ | 89.07±8.05# ▲ |
| 48 h postinjury | 10 | 36.89±4.01 | 66.92±4.98* △ |
| 72 h postinjury | 10 | 30.59±2.97* △ | 32.75±3.52# ▲ |
| 168 h postinjury | 10 | 31.43±3.90* △ | 31.07±3.25# ▲ |
* P<0.05 and # P?.01 as compared with the normal control.
△ P<0.05 and ▲ P?.01 as compared with the sham-operated group value.
The water content of the grey and white matters remarkably increased 30 minutes postinjury and continued to increase afterwards. The increase was more marked in the 6th hour and the water content approximated the control level in the 168th hour after injury (Table 2).
The pathological findings of the brain tissue were that the space surrounding the cerebral microvessels was markedly enlarged, the neurons were swollen and the Nissl's bodies decreased in number or vanished totally. These findings were most prominent in the 6th hour postinjury.
Table 2 Water content of the brain tissue in the periphery of
the contused region of the left parietal lobe
| Groups | n | Brain water content (%) |
| Grey matter | white matter |
| Normal control | 10 | 78.34±0.48 | 77.47±0.51 |
Sham-operated | 10 | 78.72±0.49 | 77.58±0.44 |
| Brain-injured | | | |
| 0.5 h postinjury | 10 | 80.14±0.37# ▲ | 78.19±0.50# ▲ |
| 1 h postinjury | 10 | 80.40±0.46# ▲ | 78.35±0.43# ▲ |
| 2 h postinjury | 10 | 80.56±0.62# ▲ | 78.42±0.54# ▲ |
| 3 h postinjury | 10 | 80.78±0.52# ▲ | 78.67±0.53# ▲ |
| 6 h postinjury | 10 | 81.51±0.57# ▲ | 78.79±0.34# ▲ |
| 24 h postinjury | 10 | 80.95±0.54# ▲ | 78.99±0.49# ▲ |
| 48 h postinjury | 10 | 80.85±0.38# ▲ | 79.04±0.52# ▲ |
| 72 h postinjury | 10 | 80.40±0.64# ▲ | 78.52±0.49# ▲ |
| 168 h postinjury | 10 | 79.07±0.36 | 77.48±0.41 |
# P<0.01 as compared with the normal control value.
▲ P<0.01 as compared with the sham-operated group value.
DISCUSSION
The metabolic dislocation of neurotransmitters in the neurons after brain injury and consequent ischemia is responsible for a large amount of release and accumulation of monoaminergic neurotransmitters such as norepinephrine, 5-HT and DA in the injured area and its surroundings. They can enter the wall of cerebral vessels and its surroundings to impair the functions of blood brain barrier and result in secondary cerebral damage eventually.4 ，5 Akiguchi et al6 reported that a large amount of fluorescent products of norepinephrine and DA piled up in the inner membrane of cerebral vessels after cerebral ischemia in the rat. Immunohistochemical assay showed that 5-HT markedly increased in the cerebral vascular wall 6 to 24 hours after cerebral trauma to exacerbate traumatic cerebral edema.7
These findings indicate that the accumulation of monoaminergic neurotransmitters in the cerebral vascular wall in the injured area and its surroundings may be responsible to induce secondary cerebral damage after cerebral trauma and ischemia. Nevertheless, all these reports dealt with the problem only qualitatively. In our study, the levels of 5-HT and DA in cerebral microvasculature were determined with HPLC-ECD in rats after brain injury and it was found that both 5-HT and DA significantly increased from 30 minutes to 6 hours postinjury. This finding further confirms the increase of 5-HT and DA in the early stage of brain injury quantitatively.
It is considered that there are 3 possible factors to result in the increase of 5-HT and DA in the cerebral microvasculature after brain trauma, traumatic stimulation to the neurons to release excessive amount of 5-HT and DA, impairment of blood brain barrier to induce the entrance of 5-HT and DA from blood stream to brain tissue and opening of neuronal Ca++ channel to result in an increased synthesis of 5-HT and DA. The changes of 5-HT and DA metabolism due to brain injury result in the accumulation of 5-HT and DA in the contused region and its periphery. Eventually, 5-HT and DA enter the cerebral microvasculature in large quantity.
It was shown that 5-HT elevated the permeability of pail microvessels of the frog brain evidently and a large amount of fluorescent tracers penetrated through the blood brain barrier to enter the brain tissue in association with a marked increase of brain water content.8 5-HT can bind with the corresponding receptors to induce secondary damage to the brain. It was reported9 that 5-HT possessed dual function: it can constrict the cerebral vessels to reduce regional blood flow and aggravate ischemia and anoxia of the neurons and it also can open the tight endothelial junction and increase permeability to induce vasogenic cerebral edema. It was found in our study that 5-HT increased in the cerebral microvasculature along with the increase of water content of the brain tissue accompanied with corresponding pathological changes. These findings indicate that the increase of 5-HT can induce the opening of blood brain barrier, which is one of the important factor to induce cerebral edema postinjury. The relationship of the increase of DA after brain injury with the occurrence of secondary cerebral damage is not clear at present and awaits further investigation.
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