2. Introduction Cerebral vasospasm (CVS)
→ reversible contraction of cerebral vessels in response to physiological and mechanical stimuli following subarachnoid hemorrhage
→ Immunoreactive, inflammatory and mechanic mechanisms take part in provoking long term contraction and inhibiting vasodilatation. This process continues with depression of smooth muscle metabolism.
→ We hypothesized that, D-Penicillamine can depress this stimulation process

3. Introduction D-Penicillamine
→ D-Penicillamine catalases a variety of metals including copper, lead, iron and mercury. Therefore it has been used treating heavy metal intoxication, Wilsons disease, but also rheumatoid arthritis
→ Artificial myasthenia gravis may occur during the treatment of auto immune diseases especially rheumatoid arthritis with D-Penicillamine
→ Just like in myasthenia gravis, D-Penicillamine generates auto-anti-receptors against post-synaptic alpha-acetylcholine-receptors and causes as a result artificial myasthenia gravis like symptoms
→ We planned to investigate the effects of D-Penicillamine on CVS in rabbits, who underwent autologous blood injection into the cisterna magna in order to induce SAH

4. Basilar angiography only
Methods
Treatment groups
→ The study group consisted of 30 rabbits which were divided into 4 groups
Group I
N = 5
Basilar angiography only
Group II
N = 5
Basilar angiography following intra peritoneal penicillamine application for 3 weeks
Group III
N = 10
Induction of artificial SAH by injecting autologous blood into cisterna magna
Group IV
N = 10
Induction of artificial SAH following intra peritoneal penicillamine application for 3 weeks.
→ Subjects were kept in a room with an avarage temperature of 27°C. At the end of the third week, the subjects were anesthetized with 2mg/kg xylazine and 12,5mg/kg ketamine and intubated
→ After canulation of the central ear artery with a 22 gauge catheter, blood pressure, pulse and mean arterial pressure were monitored
→ The right femoral artery was canulated at the inguinal level, and vertebral angiography was performed with 5cc celtrevist at minutes. During this process the ventilator was arranged to be at pCO mm Hg

5. Methods Treatment Artificial SAH
→ All subjects were sacrificed following the 4th (180 minutes treatment) basilar artery angiography and the basilar artery was dissected under microscopic view after immediate craniotomy
→ The luminal width of the basilar artery was measured in pixels and converted into mm. (1 pixel equals 0,264mm)
→ Group II subjects, different from the 1st group, underwent intra-peritoneal D-Penicillamine injection daily (10mg/kg)
→ In all subjects, muscle biopsy from the right orbicularis oculi muscle was performed, in order to detect signs of myasthenia gravis.
Artificial SAH
→ in group III + IV, following basilar artery angiography, rabbits were placed in Trendelenburg position and the hair around the nuchal region was washed. Then 2ml of non-heparinized autologous arterial blood was withdrawn from the ear artery and percutaneously injected into the cisterna magna with 23 gauge needle
→ After withdraw of 2cc cerebrospinal fluid (CSF), 1cc/min blood was injected. The rabbits were positioned in supine position for 15 minutes in order to facilitate settling of the blood around the basilar artery

6. Methods Treatment of SAH subjects Statistics
→ Group IV: The subjects were injected once a day with intra-peritoneal D-Penicillamine (10mg/kg) for 3 weeks.
→ 2 rabbits from the 3rd group and 1 from the 4th group could not be evaluated, due to decerebration after blood injection into the cisterna manga
→ One subject from the 3rd group could not be evaluated due to exitus letalis during angiography
→ EMG was only performed in rabbits that underwent D-Penicillamine treatment.
Statistics
→ The student T- test was used for statistical analysis and p-value was considered as significant at 0.05.

7. Methods Evaluation criteria
→ Cerebral vasospasm by basilar angiography.
→ Vascular degeneration by electron-microscopy examination of the basilar artery, free O2 radicals by investing vascular wall superoxide dismutase (SOD) and malondialdehyde (MDA) levels
→ Myasthenia gravis by electromyography (EMG) and electron-microscopy examination of the orbicularis oculi muscle

8. Results Basilar artery radius Group III Group IV 0 min 30 min 180 min

9. Results
EMG (orbicularis oculi muscle)

10. Results
Electron microscopy

11. Results
Electron microscopy

12. Results
Electron microscopy

13. Results
Electron microscopy

14. Conclusion D-Penicillamine in SAH-induced CVS
→ Significant decrease of free oxygen radicals in D-Penicillamine treated groups.
→ 10% vessel dilation at the 180th minute in group IV, compared to group III. This can be explained by the decrease in free O2 radicals and a possible anti-inflammatory effect
→ Myasthenia gravis did not develop in our subjects
→ EMG and electron-microscopy examination of orbicularis oculi muscle in D-Penicillamine treated groups were found to be normal

15. Conclusion
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