| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Eur J Cardiothorac Surg 2005;28:467-472
© 2005 Elsevier Science NL
Original articles |
a Department of Cardiovascular Surgery, Istanbul University, GATA, Haydarpasa Military Training Hospital, Istanbul 84390, Turkey
b Department of Cardiovascular Surgery, Gata Military Training Hospital, Ankara, Turkey
c Department of Neuropathology, Istanbul University, Istanbul, Turkey
Received 12 April 2005; received in revised form 8 June 2005; accepted 13 June 2005.
* Corresponding author. Tel.: +90 212 534 00 00; fax: +90 212 534 22 32. (Email: dr_murat_basaran{at}yahoo.com).
Abstract
Objective: This study was aimed to test the hypothesis that the combination of heat stress and early ischemic preconditioning (IP) applied before aortic occlusion would be protective against spinal cord ischemic injury. Methods: Thirty Whister–Albino rats were randomly divided into three groups. In group 1 (n=10), aorta was clamped just distal to the left renal artery and above the iliac bifurcation for 45min. Group 2 (n=10) had 5min of transient aortic occlusion and 30min later underwent an additional 45min. In group 3 (n=10), animals were heated to 41°C and maintained at this temperature for 15min. Twenty-four hours later, this hyperthermia pretreated group underwent the same early IP model of aortic occlusion. Neurologic status was assessed on postoperative 24 and 48h by using the 15-point neurologic performance scale. Spinal cords were harvested for histopathological grading (1–4) and evaluated for the presence of heat shock protein-ubiquitin staining. Results: At 24 and 48h, the mean neurologic performance scores of the group 1 were found to be significantly lower than those of groups 2 and 3. Although the neurologic assessment of rats performed on the 24h did not reveal statistically significant difference between groups 2 and 3 (P=0.069); on 48h, the mean neurologic scores of the group 3 were significantly higher than those of group 2 (P=0.005). At 48h, a delayed neurologic deterioration was seen in groups 1 and 2 when compared to the results obtained at 24h. Histologic evaluation correlated well with the neurologic outcome with the least cellular damage in group 3. There were six rats with ubiquitin expression and this was detected only in animals pretreated with sublethal heat stress. Conclusions: An early IP model with a short reperfusion interval does not give the minimal required time for the HSPs expression and is associated with a delayed neurologic deterioration. Neuroprotection provided by heat stress combined with an early IP model lasts up to 48h and heat shock protein-ubiquitin induction may be responsible in this phenomenon.
Key Words: Hyperthermic ischemic preconditioning • Spinal cord ischemic injury • Early ischemic preconditioning
1. Introduction
Paraplegia is a devastating complication that may result from operations requiring transient occlusion of the thoracoabdominal aorta. Despite considerable improvements in surgical technique, Svensson reported a 16% incidence of neurologic injury in patients having major aortic interventions [1]. Although numerous experimental and clinical investigations have been performed to reduce the incidence of paraplegia, no reliable method has been developed that totally prevents spinal cord ischemic injury.
Ischemic preconditioning (IP), in which brief periods of noninjurious ischemia increase tolerance to subsequent lethal ischemic insult, was first mentioned by Murry and co-workers [2]. Later, several authors have investigated its effects and IP has been found to be protective in many organs including heart, brain and spinal cord [3–5]. The proposed mechanisms by which IP reduces neurologic injury included preservation of adenosine triphosphate (ATP) stores, activation of AI adenosine receptors, activation of potassium channels and induction of heat shock proteins (HSPs) [2,6–8]. Nowadays, the main focus of attention has been directed to delayed protective effects of IP, termed the ‘second window of protection’, which appears more than 24h after the initial insult [7,9]. There is a growing body of evidence suggesting that this delayed effect of IP is related to the new synthesis of proteins, termed HSPs, that are crucial for the maintenance of cellular integrity under unfavorable conditions [10,11]. In these experimental models, the investigators have generally used a late IP model in which the reperfusion interval between the IP and the subsequent lethal ischemic insult was several days. Although recent findings suggested that shorter reperfusion intervals also reduce ischemic spinal cord injury [3,12]; to date, only a limited number of animal studies have investigated the neuroprotective role of HSPs in an early IP model and we believe that this model of preconditioning does not give the minimal required time for the HSPs expression.
It has been demonstrated that whole-body hyperthermia can also lead to the induction of HSPs which are able to increase the resistance of the tissues to ischemic and non-ischemic insults [13]. In this experimental study, we aimed to test our hypothesis that an early IP model with a short reperfusion interval would protect transiently against spinal cord ischemic injury; and, the combination of early ischemic preconditioning with whole-body hyperthermia would provide the delayed anti-ischemic effect by way of HSPs expression and improve the clinical outcome.
2. Material and methods
The handling and care of all animals were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The animal protocol was approved by the Institutional Animal Research Committee and conducted with the financial aid of Scientific Research Committee (BEKADEB) of the Istanbul University.
2.1 Experimental protocol
A total of 30 Whister–Albino rats of either sex, weighing 350–450g, were used in the study. They were allowed free access to food before the experimental procedure and all were neurologically intact prior to anesthesia.
General anesthesia was induced by intraperitoneal injection of sodium pentobarbitale (50mg/kg). Rats did not receive mechanical ventilatory support throughout the procedure and allowed to breathe spontaneously with an oxygen face-mask. Intramuscular cephalosporin was administered for antibiotic prophylaxis before the skin incision.
The rats were placed in the supine position and following local anesthesia, a short incision was made in the neck region. Care was taken to avoid left superior vena cava, and the left jugular vein was cannulated with a 24-gauge venous catheter for intravenous injection of drugs and fluids. We placed a 24-gauge catheter of appropriate length into the left common carotid artery and the proximal mean aortic blood pressure (PAP) was monitored with a pressure transducer. Temperature probe was inserted into the rectum and core body temperature was continuously monitored during the entire procedure. Body temperature was maintained at 37±1°C by using a heating pad.
The peritoneal cavity was then exposed through a vertical incision and the infrarenal abdominal aorta was exposed. The rats were randomly divided into three groups. In group 1 (control rats, n=10), following the intravenous administration of heparin (150IU/kg), microvascular clamps were placed just distal to the left renal artery and above the iliac bifurcation for 45min. In group 2 (n=10), an early IP model was applied by clamping the aorta proximally and distally for 5min before 45min of aortic clamping with a reperfusion interval of 30min between. In group 3 (n=10), after the induction of general anesthesia, the rats were heated to 41°C with a heating lamp and maintained at this temperature for 15min. Then, they were allowed to recover from the anesthesia and cool to normal core body temperature in their cages. On recovery, animals were re-hydrated with normal saline (intraperitoneal, 10ml/kg) and used for experiments 24h later. The hyperthermic ischemic preconditioning group underwent the same described early IP model of aortic occlusion. The aortic isolated ischemic segment pressure (IASP) was monitored with a 24-gauge catheter throughout the occlusion in all groups and it was kept less than 15mmHg. Arterial blood gases (PaCO2 and PaO2) and pH were measured at baseline, during aortic occlusion and after reperfusion. Arterial oxygen saturation was also followed continuously by pulse oximetry during the peri- and postoperative periods until the animals recover from the anesthesia to detect hypoxia. After the completion of the surgery, the abdominal incision was closed in two layers with silk suture and all animals were allowed to recover in individual cages at 25°C.
2.2 Neurologic evaluation
All animals were closely monitored after the procedure. Neurologic status was assessed on postoperative 24 and 48h by using the 15-point spinal cord performance scale (Appendix A) [13,14]. The neurologic examinations were done in a blinded fashion.
2.3 Histopathologic examination
The animals were sacrificed at the end of last neurologic assessment. All rats were anesthetized with an intraperitoneal injection of sodium pentobarbitale (50mg/kg) and perfused intracardiacally with 100ml of 0.9% normal saline solution, followed by 750ml of 4% paraformaldehyde in 0.1M phosphate buffer solution (PBS, pH: 7.44). Following perfusion, whole spinal cords were dissected and removed. The entire spinal cord specimen was postfixed for 24h in the same fixative and embedded in paraffin blocks for later sectioning. At the end of 120h, 5µm thickness sections were obtained from the lumbosacral spinal cord and stained with hemotoxlin and eosin (HE) for light microscopic examination. A neuropathologist who was blinded to experimental procedure performed the histologic evaluation in the light microscopy for the presence and degree of cellular degeneration, cytoplasmic eosinophilia, loss of cell membrane integrity, chromatin condensation and number of intact motor neurons in the gray matter (per high-powered field). The grade of ischemic injury and histologic scores were determined by using our histopathological grading scale: 0 point=severe ischemic injury with eosinophilic neuronal degeneration, loss of cell membrane integrity and vacuolization; 1 point=moderate ischemic injury with eosinophilic neuronal degeneration, loss of cell membrane integrity and vacuolization; 2 point=mild ischemic injury with eosinophilic neuronal degeneration, loss of cell membrane integrity and vacuolization; 3 point=no injury.
2.4 Immunohistochemistry for ubiquitin
Five-micrometer sections obtained from the lumbosacral region were mounted onto poly-L-lysine coated slides. The sections were immersed in 0.3% H2O2 for 15min and washed with PBS. The sections were then incubated in anti-Ubiqutin polyclonal antiserum from rabbit for 1h. After the primary incubation and three rinses in PBS, sections were incubated in biotinylated horse anti-mouse IgG for 10min. Following the incubation in substrate chromagen solution for 10min, all sections were washed in PBS and distilled water, mounted glycerol and examined later under electron microscopy. The spinal cord sections stained positively for ubiquitin was assessed and compared among groups.
2.5 Statistical analysis
Data are expressed as mean±SD. Statistical analysis was performed by one-way analysis of variance test with the post-hoc Tukey honestly significant difference for comparison of physiologic variables between groups. The comparison of neurologic and histologic scores was performed with Kruskal–Wallis test, whereas differences between two groups were analysed with Bonferonni-adjusted Mann–Whitney U-test. The comparison of neurologic scores within a group was performed with paired-sample t test. The correlation between neurologic and histologic scores was investigated using Spearman's rank correlation analysis. Differences were considered statistically significant for a P-value of <0.05.
3. Results
Mortality for the 45min of occlusion was 6.6% (2/30). One rat in group 1 and another rat in group 2 died after the experimental procedure. The physiologic variables recorded during the experimental procedure are presented in Table 1 . The rectal temperature did not differ between groups throughout the operation period (P=0.67). No significant difference was noted among groups in terms of mean IASP during aortic occlusion (P=0.6). During the occlusion and reperfusion periods, we tried to keep PAP at 75±5mmHg. To overcome systemic acidosis and possible blood pressure depression, we used a mixture of combination of 0.9% normal saline solution and sodium bicarbonate. There was no significant difference among groups in the amount of infusions given during the entire procedure (P=0.45). Arterial blood gases were similar in all groups and hypoxia was not detected during the peri- or postoperative period.
|
|
|
Examination of the spinal cords of the groups 1 and 2 revealed moderate or severe neuronal damage with cellular degeneration and cytoplasmic eosinophilia (Fig. 1 ). The gray matter of the rats pretreated with whole-body hyperthermia showed no or only minimal injury. Lumbosacral spinal cord tissue from group 3 demonstrated almost preserved gray matter architecture with highest viable motor neuron cell number per high-powered field (Fig. 2 ). The mean number of intact, heathy motor neuron cells in groups 1–3 were 11.3±1.8, 14.1±2.4 and 19.2±1.4, respectively (groups 1 vs 2, P=0.015; groups 1 vs 3, P=0.01; groups 2 vs 3, P=0.03) (Fig. 3 ).
|
|
|
|
The results of this study revealed that hyperthermic IP provides protection against spinal cord ischemic injury. Although an early IP model with a reperfusion interval of 30min reduces neurologic injury, this neuroprotection is transient and a delayed neurological deterioration was noticed in this group of animals.
The optimal method for the protection of spinal cord during the aortic occlusion has been debated since the begining of modern aortic surgery. A large variety of techniques have been advocated, but their utility in clinical practice is still controversial. Several studies have investigated the effects of ischemic preconditioning that has been found to be protective in many organs including heart, brain and spinal cord [3–5]. Although the beneficial effects of both early and late IP models in reducing spinal cord injury have been confirmed, the reperfusion interval differed significantly in the experimental models and there is no still an established consensus about the optimal duration of reperfusion between two ischemic insults.
Recent investigations that have been focused on the protective effects of IP revealed a delayed anti-ischemic effect of IP which appears more than 24h after the initial insult [7,10]. In these experimental models, the investigators have generally used a late IP model with a reperfusion interval of 48h and have emphasized the importance of HSPs synthesis as a mechanism of neuroprotection [15,16]. Improved understanding of mechanisms of ‘second window protection’ has elucidated the role of HSPs that are a subgroup of proteins playing an important role in the acquisition of ischemic tolerance in neuronal cells. They are crucial for the maintenance of cellular integrity under unfavorable conditions and act as a ‘molecular chaperone’ by preventing the degradation of denatured proteins.
HSPs induction may be achieved by various insults including ischemia, trauma, hyperthermia and drug administration [13]. In this experimental model, we aimed to induce their synthesis by thermal pretreatment. The molecular aspects of neuroprotection provided by heat stress has been demonstrated; but, to date, the optimal time interval between sublethal stress and the subsequent insult is not clearly defined. Some authors demonstrated that the functional improvement provided by hyperthermia was greatest when heat stress was applied 18–24h before the ischemic event [17,18]. In this experimental study, heat stress applied 24h before the ischemic insult is associated with tissue salvage and prevents spinal cord ischemic injury by the induction of HSPs.
Xia demonstrated that neurons in the gray matter is the preferential target of the heat shock response and suggested that the heat shock response might have a therapeutic implication for protection against spinal cord injury [19]. In contrast to this finding, in his experimental study performed in hyperthermia pretreated rabbits, Manzerra [20] reported that the heat shock response occured in glial cells, not in the large motor neurons. In our study, the motor neuron cells appear to be the preferentitial target of heat stress as demonstrated by immunohistochemistry. However, the interpretation of the results from different studies is difficult because of the differences in animal species and experimental models; but, we know that different tissues have varied HSPs synthesis potential after preconditioning and the time required for the induction of HSPs differs between tissues [21].
The trend toward functional and histologic preservations with sublethal heat stress became statistically significant at 48h of aortic occlusion, at which time both control and early IP groups had lower neurologic scores than hyperthermia pretreated group. Our findings also demonstrated that ischemic neuronal injury is an ongoing process and the detection of normal motor function during the early post-experimental hours does not mean the absence of significant histopathologic injury. In addition, although six out of 10 rats demonstrated HSPs expression, superiority of neurological scores was evident in all members of the hyperthermia pretreated group. Thus, to date, only a limited number of studies have investigated these complex interactions and there must be also additional protective effects of heat stress other than HSPs induction. We believe that improved understanding of the exact mechanisms would lead us to develop new methods for the prevention of ischemic neuronal injury.
Although necrosis is the main cause of ischemia induced neurologic injury, recent reports demonstrated that apoptosis has also an important role in this process [22]. It has been demostrated that heat shock response exert also an anti-apoptotic activity in various mammalian tissues [23]. Within the first few days following transient spinal cord global ischemia the motor neurons undergo apoptosis and heat stress may modulate the processes that have been attributed in the pathogenesis of spinal cord ischemia leading to cellular necrosis and apoptosis, such as excitatory amino acid excitotoxicity, free radical overproduction, and nitric oxide overproduction. However, in this study, we did not aim to evaluate the roles of apoptosis, inflammatory response and various cytokines in the development of ischemic spinal cord injury.
The role of this family of protein in an early IP model is a subject of considerable debate. Although previous reports have shown beneficial effects of early IP model [12]; we hypothesized that a model with short reperfusion interval would not give the required time for the expression of HSPs and could not provide the delayed neuroprotective effect of IP. In their experimental studies, Matsuyama [15] and Sakurai [16] used a long reperfusion interval and revealed a positive correlation between HSPs expression and neuroprotection. In another study reported by Cizkova [24], an at least 6min of sublethal interval of preconditioning is required for a potent neuronal HSPs induction. In our study, we used a 5min of early IP and did not find any correlation between HSPs expression and neuroprotection. Therefore, we agree with Zvara [12] who suggested that additional mechanisms other than HSPs may play important roles in this type of early IP model. Our study demontrated that although an early IP model has protective effects, this neuroprotection is not related to the synthesis of HSPs. However, our results differ from those of Zvara and the protective effects provided by this model is transient and do not last up to 48h. Bimodal HSP expression is an important cause of the ‘second window of protection’ provided by IP [11] and a time interval between two insults is required to permit enough time for the production of these stress proteins.
Therefore, from these data, we concluded that these molecular chaperons are particularly important for the continuation of protective effect and their absences may lead to a delayed neurological deterioration.
There are some limitations to this study. As with previous studies [4,25], the present model was performed by placing microvascular clamps at the level just distal to the left renal artery and above the iliac bifurcation. Although the period of ischemia was well tolerated in all animals, the model used in this study requires at least 45min of aortic occlusion which may be associated with a higher intraoperative mortality rate. To overcome this problem, the animals undergoing the experimental procedure required close monitorization and prompt intervention with intravenous fluid and drug administration. In addition, the sacrifice of animals at the end of 48h, not at specific time points of follow-up, did not provide us enough data for the progression of neuronal injury.
The results of our study showed that hyperthermic ischemic preconditioning provides neuroprotection which lasts up to 48h. There is a positive correlation between heat shock protein-ubiquitin induction and the delayed neuroprotection provided by heat stress. An early IP model provides protection only during the early post-experimental period and is not associated with the delayed neuroprotective effects of preconditioning. Thus, in an early IP that does not give the minimal required time for the expression of HSPs, the role of HSPs is controversial.
Appendix A. Spinal cord performance scale (adapted from Zhang and Lemay) [13,14]
|
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
