Xi'an 710033, China

Keywords: spinal cord; ischemic-reperfusion injury; hyper baric oxygenation; ischemic tolerance; rabbits
CLC number:R651.2 Article ID: A
Document code： 1000-2790(20 00)04-S0055-03

INTRODUCTION

Paraplegia is a severe complication ofdesendingthoracic aneurysm (TA) and thora coabdominal anerysm(TAA) repair^[1] .This complication is caused by spin al cord ischemia secondary to aortic clamping. Manymethods and drugs have been suggeste d to protect against ischemic spinal cord injury^[2-4] but the efficacy is still under dispute. Therefore, it is necessary to find some effectivemethods or dru gs to effectively protect against spinal cord ischemic injury.
In 1990, Kitagawa et al^[5] found “ischemic tolerance” phenomenoni n the brain. Matsuyama^[6] proved that this phenomenon could also beinduced in the spinal c ord. But it seems that this phenomenon only provides a good modelfor the study of the mechanisms of cerebral or spinal cord ischemic injury. Recently, anumber of studies demonstrated that some chemicals could induce the tolerance againstischemia in brain^{[ 7-9 ]} . But these chemicals can't be put into clinicalapplication due their toxicity.
Hyperbaric oxygen (HBO) has been reported to have protective effects on cerebralischemic injury when it was used after ischemia^[10,11] . HBO, which is nowin b road clinical use, represents a safe way of generating reactive oxygen species. IfHBO can possibly induce tolerance against ischemia in spinal cord, HBO, as a safepreconditioning way, should be easily
accepted in clinical practice. The ai m of the present study was to determine whether theischemic tolerance could be induced in the spinal cord in rabbits when they werepretreated with HBO.

METHODS

23 New Zealand white rabbits (2.1±0.3)kgwere randomly assigne d to three gro ups: Control group (n=8), HBO-1 group (n=8) and HBO-2group (n=7).

Rabbit spinal cord ischemia model

The rabbits were anesthetized with h alothane. In all the animals a 24-gauge cat h eter was inserted into the ear artery tomeasure the proximal blood pressure (MA P_proximal ), and a second catheter wasinserted into the left femoral arter y to measure the distal blood pressure (MAP_distal ).Rectal temperature was maintained at 38.0～39.0℃ during the experiment. Arterial bloodwas sampled for the determination of PaO₂ , PaCO₂ , pH and plasmaglucose.
The abdominal aorta was exposed at the level of the renal arteries through a med ialincision. Small diameter plastic tubing was placed around the aorta just dis tal to theleft renal artery. The ends of the tubing were threaded through a sma ll plastic buttonand then through a plastic tube of a larger diameter, forming a snare ligature. Aorticocclusion was performed by pulling and clamping the sma ll tube around the aorta. Ischemialasted for 25 min. At the end of the ischemic period, the tubing was released to restorethe flow through the aorta, and the abdominal wall was closed with wound clips. Thenanimals were put back to their home cages and observed for 2 days. Before occlusion, IVheparin 500 U was admin istered.

Experimental protocol

The rabbits in the HBO-1 group received1 h of hyperbaric oxygenation at 2.5 a t mosphere absolute (ATA) in 100% oxygen every dayfor 3 days in an animal hyperba ric chamber. The rabbits in the HBO-2 group received thesame HBO as that in HB O-1 group everyday for 5 days. 24 hr after the last pretreatment ofHBO, animal s were induced to spinal cord ischemia. The control group was treated in thesam e manner as the HBO treated group except that no hyperbaric oxygen was applied.

Neurologic and histopathologic evaluated

At 4, 8, 12, 24 and 48 h afterreperfusion, the rabbits were neurologically asse s sed by an observer who was unaware ofthe groups by using the modified Tarlov Cr iteria^[12] : 0 for no voluntaryhindlimb function; 1 for movement of join ts perce ptible; 2 for active movement butunable to stand; 3 for the ability to stand bu t unable to walk; 4 for complete recoveryof hind limb function. After the compl etion of the neurologic deficit scoring at 48 h,the animals were reanesthetized . Transcardiac perfusion and fixation were performed withheparinized saline, fol lowed by 10% buffered formalin. The spinal cord (L_5-7 )was removed and em bedded in paraffin. Coronal sections of spinal cord were cut at athickness of 6μm an d stained with hematoxylin and eosin. Neuronal injury was evaluatedat a magnific ation 400× by an observer who was unaware of the groups. Ischemic neuronswere identified by cytoplasmic eosinophila with loss of Nissl substance and by the presence of pyknotic homogenous nuclei. In each slice, normal neurons in the anter iorspinal cord were counted.

Data analysis

The hind limb motor function, and thenumbers of normal neurons in the anterior spinal cord were analyzed using a nonparametricmethod (Kruskal -Wallis test). Changes in MAP, HR, rectal temperature, pH, PaO₂ ,PaCO₂ and glucose were co mpare d by using t test. P value less than 0.05 wasconsidered statistically s ignificant.

RESULTS

There were no significant differencesamong the groups in MAP, rectal temperatur e, pH, PaO₂ , PaCO₂ andplasma glucose variables, except for HR. All animals surv ived to the final neurologicscoring (48 h after reperfusion). Histopathology of the lumbar spinal cord was examined in21 animals.

Time course of neurological outcome

The neurological status at 4, 8, 12 ,24 and 48 h in the HBO-2 group was significantly better than that in the cont rol group.At 24 and 48 h, the neurological status in the HBO-2 group was signi ficantly better thanthat in HBO-1 group. There was no significant difference i n neurological status betweenthe HBO-2 and HBO-1 groups throughout the assess ment except at 48 h (see Fig 1).

Fig 1 The time course of neurologicaldeficit scores
^a P<0.05 vs control,^b P<0.01 vs control.

Neurological outcome

The neurological outcome in both HBO-1and HBO-2 groups were better than that in control group (P<0.05). The neurologicaloutcome in HBO-2 was better than that in HBO-1 group (P<0.05, Fig 2).

Histopathological outcome

The normal neurons inthe anterior spinal cord of HBO-2 group were more than th ose of control and HBO-1 group(P<0.05). The number of normal neurons in th e an terior spinal cord of HBO-1 group wassimilar with that of control group (Tab 1 ).

Fig 2 The neurological outcome at 48 hafter reperfusion
^a P<0.05 vs control,^b P<0.05 vs HBO-1.

Tab 1 The numbers of normal neurons inthe anterior spinal cord 48 h after reperfusion

DISCUSSION

The present study for the first time demonstrated that the hyperbaric oxygenper formed before spinal cord ischemia could induce ischemic tolerance. 5-day HBO ( 1 hper day) preconditioning induced stronger ischemic tolerance than the 3-day HBOpreconditioning.
Since the ischemic tolerance in the brain was found in 1990, many studies have b eenperformed to investigate the mechanisms of this phenomenon^{[6-9 ]} . Because the ischemic tolerance induced by a brief period of sublethal ischemia is difficu lt toperform in clinical practice, studies have been conducted to find a drug o r chemical toinduce ischemic tolerance. Although a number of papers had reporte d that some chemicalscould induce this phenomenon in brain, those substances ha d not been applied to theclinical practice for their toxicity^[7-9] . H yperbaric oxygen is a safe andeasily performed therapy in clinical practice, and its pro tective effect on cerebralischemia has been evidenced before^[10,11] . F ree oxyg en radical (FOR),which can be generated by HBO, has been proved to be able to i nduce ischemic tolerance ingerbil hippocampal neurons^{[13 ]} . Therefore we hypot hesized that HBO couldinduce ischemic tolerance in spinal cord ischemia. The re sults of this study have provedit.
The mechanism, by which HBO preconditioning induces ischemic tolerance in spinalcord, is not clear. Based on the confirmed findings that HBO increases the amou nt ofoxygen free radicals generated in tissues^[14,15] , and the oxidati ve stress can induce tolerance against ischemia^[13] , we suggest that the generation of oxygen free radicals and lipid peroxides by hyperbaric oxygenation may be the major mechanism by which HBO preconditioning induces ischemic tolerance. But more studiesare needed to further to clarify this.
In conclusion, hyperbaric oxygen, a wildly used therapy in clinical practice, ca ninduce ischemic tolerance in spinal cord of rabbits if pretreated before ische mia.

(Editor YUAN Tian-Feng)

Foundation item: This study issupported by a grant from National Natural Science Foundation No. 39670699
Biography:Dong Hai-Long (male, born on July 1, 1974, i n the City of Baoji, ShaanxiProvince) graduated from Fourth Military Medical Un i versity in 1993 and is now studyingfor Master's degree. His supervisor is Prof XIONG Li-Ze. Tel.(029) 3375344 E-mail.mazuike@ fmmu.edu.cn

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Received date:1999-09-02 ; Reviseddate:1999-12-16 (DONG Hai-Long, XIONG Li-Ze, ZHU Zheng-Hua)