a b s t r a c t

Background: This experimental study was performed to investigate the Neuroprotective effects of progesterone on spinal cord ischemia in rabbits.

Methods: Eighteen female New Zealand white rabbits were used in this study. Rabbits were randomized into 3 groups. Spinal cord ischemia was induced by clamping the abdominal aorta from a point just inferior to the left renal artery to the aortic bifurcation for a period of 30 minutes. Group 1 served as the control group, and groups 2 and 3 received intraperitoneal progesterone immediately after the onset of reperfusion, at a dose of 8 mg/kg. Two hours after reperfusion, the animals in group 1 were killed. Four hours after reperfusion, the animals in group 2 were killed, and 6 hours after reperfusion, the group 3 rabbits were killed. Spinal cords were removed and fixed in 10% formalin in a phosphate buffer. Neuronal injury was evaluated by a pathologist who was blinded to the treatment groups, and 5 sections per animal were evaluated. The number of intact large motor neuron cells in the ventral grey matter region was counted.

Results: The analysis revealed that the average mean arterial pressure for group 1 was significantly higher than that for group 2, and the mean sacrificed pressure value for group 1 was significantly higher than that for group 3 (P b .05). The number of intact neurons in group 1 was significantly lower than the number of intact neurons found in both groups 2 and 3 (P b .05). No other statistically significant differences were found between the groups.

Conclusion: The findings from the present study indicate that progesterone effectively protects the spinal cord tissues against ischemic damage in the setting of decreased perfusion.

(C) 2013

Introduction

Spinal cord ischemia is a relatively Rare condition, affecting approximately 12 in 100000 people in the general population [1]. The prognosis for spinal cord ischemia tends to be very poor. There is a high risk of death, both during the acute phase of infarction and over the long term. It has been estimated that spinal cord infarction accounts for approximately 1% of all strokes [2].

Available treatment options are, unfortunately, limited. The standard drug therapy is aspirin [3]. However, there are no clear guidelines for the treatment of spinal cord ischemia. Given the uncommon nature of the disorder, frequent delays occur in making

* Corresponding author. Tel.: +90 232 244 44 44 1218; +90 506 683 08 70 (Mobile tel).

E-mail addresses: [email protected] (N. Vandenberk), [email protected] (E.E. Unluer), [email protected] (N. Gokmen), [email protected] (I. Yurekli), [email protected] (E. Okmen), [email protected] (O. Yilmaz), [email protected] (S. Yigit),

an accurate diagnosis. Animal investigations have shown some benefit with certain agents, such as prostaglandins, nimodipine, naloxone, adenosine, and magnesium, but there have been no prospective clinical studies evaluating their efficacy in treating spinal cord ischemia. One currently untested therapeutic agent for this condition is progesterone, which has been shown by several research groups to possess potent neuroprotective properties in Experimental models of brain injury and ischemia [4-7]. When progesterone has been used as a neuroprotective agent in placebo- controlled traumatic brain injury clinical trials, only limited side effects have been reported in association with its use. Moreover, the feasibility of administering high-dose intravenous progesterone in other fragile patient populations further underscores the acceptabil- ity of this drug’s safety profile [8].

Progesterone has a substantial window of therapeutic efficacy and can be given up to 24 hours after injury and still have beneficial effects in animal models and cases of traumatic brain injury [9,10]. The first successful clinical trial for the treatment of traumatic brain injury, after more than 30 years of research in the field, was published in

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2007 [10]. A phase IIa, single-center clinical trial investigating the efficacy of progesterone in the treatment of moderate-to-severe adult traumatic brain injury found that the mortality rate among patients given intravenous progesterone for 3 days postinjury was less than half that of the controls (13.6% vs 30.4%).

The objective of our study was to determine whether progesterone demonstrates a neuroprotective effect in rabbits with spinal cord ischemia.

Materials and methods

Eighteen female New Zealand white rabbits (8-12 months old), each weighing between 2.4 and 3.5 kg, were used in this study. All animals were housed under standard conditions in the Animal Research Laboratory at Dokuz Eylul University. The study protocol was approved by the animal research committee of Dokuz Eylul University. A total 18 rabbits were used, and none died during surgery.

Anesthesia and monitoring of animals

The animals were fasted for 12 hours and humanely restrained. Anesthesia was induced with 3% halothane in 100% oxygen and was maintained with 0.5% to 1.5 % halothane in a mixture of 50% oxygen and 50% room air. End-tidal concentrations of halothane and CO₂ were continuously measured with a monitor (Anaesthetic Gas Monitor Type 1304; Bruel & Kjaer, Naerum, Denmark) via nasopha- ryngeal sampling.

The retroauricular vein in the right ear was cannulated, and an infusion of 0.9% NaCl solution was started at a rate of 4 mL/kg per hour. An artery in the left ear was also cannulated to monitor arterial blood pressure and allow for arterial blood gas sampling. To monitor proximal and distal aortic pressures, catheters were placed into the aorta and the femoral arteries. Verification that the appropriate level of sedation had been reached was determined by the lack of a righting reflex and by testing the palpebral and pedal withdrawal reflexes every 10 minutes, as previously described by Wyatt et al [11]. All experiments were performed under the same conditions. Rectal temperatures were maintained at 38.5?C by keeping the animals under a heat lamp until their recovery from anesthesia.

Surgical procedures

The sedated animals continued to breathe spontaneously and were placed in the right lateral decubitus position. The skin was prepared with povidone iodine and anesthetized with bupivacaine (25% solution), and an incision was made in the flank, parallel to the spine at the 12th intercostal level. Following incision and dissection through the thoracolumbar fascia, the longissimus lumborum, and iliocostalis lumborum muscles were retracted. The abdominal aorta was exposed via a left retroperitoneal approach and mobilized just inferior to the left renal artery, where it was clamped, down to the point of the aortic bifurcation. Each rabbit was anticoagulated with

400 U of heparin before Aortic occlusion. After 30 minutes of occlusion, the catheters were removed, and the incision was closed. The animals were monitored until they fully recovered and were then returned to their cages.

Experimental design

The animals were randomly divided into 3 groups, each consisting of 6 rabbits. All of the rabbits were female. We obtained presurgical and postsurgical 2-mL blood samples from each rabbit to measure progesterone levels. Group 1 (control) animals received nothing following reperfusion. Groups 2 and 3 (treatment) received intraper- itoneal progesterone (Progynex 50 mg/mL) immediately after the onset of reperfusion at a dose of 8 mg/kg. This specific dose of

progesterone was chosen because it has been shown in multiple studies to prevent neuronal loss after brain injury and ischemia (Stein et al 2006, Roof et al 1994, Thomas et al 1999). After completion of the surgical procedures, the tube and catheters were removed, and the incision was closed. The animals were monitored until they fully recovered and were then returned to their cages. Two hours after reperfusion, the animals in group 1 were killed. Four and 6 hours after reperfusion, the animals in groups 2 and 3, respectively, were killed with intraperitoneal sodium thiopental (120 mg/kg). The spinal cords from all animals were removed and fixed in 10% formalin in a phosphate buffer.

Monitoring the physiologic parameters

During the surgical procedure, the heart rate, mean arterial pressure, and rectal temperature were continuously monitored (Biopac MP30 and Biopac BSL pro v.3.6.5; Biopac Systems, Santa Barbara, CA), in addition to the respiration rate and end-tidal CO₂ level. Following surgery, the rabbits were placed in a warming chamber, and their body temperatures were maintained at approx- imately 37?C until they were completely awake.

Determination of progesterone levels

Blood samples (2 mL) were taken from the peripheral veins of all the animals before surgery and before sacrifice to measure serum progesterone levels. Once postsurgical progesterone levels were determined, the animals were killed. Blood samples remained at Room temperature for 1 hour, until they clotted. Samples were then centrifuged for 10 minutes at 4000 rpm to obtain serum specimens. Serum specimens were stored at 4?C and analyzed within 24 hours to determine the progesterone level, which was measured according to Bar-Or et al’s colorimetric method [12].

Histopathology design

Spinal cords were removed and fixed in 10% formalin in a phosphate buffer. After fixation, transverse sections of the spinal cord at the L5 level were embedded in paraffin, cut into 5-um- thick sections, and stained with hematoxylin and eosin. Neuronal injury was evaluated at x40, x100, x200, and x400 magnifica- tions by a pathologist blinded to the treatment groups. Five sections per animal were read. We performed hematoxylin and eosin staining on a set of sections and examined them using light microscopy. The number of intact large motor neuron cells in the ventral gray matter region was counted. The observers, blinded to the experimental groupings and neurologic outcomes, examined each slide. Following hematoxylin and eosin staining, the cells were considered to be dead if the cytoplasm was diffusely eosinophilic and were considered viable if the cells demonstrated basophilic stippling.

Statistical analysis

For statistical evaluation, we used the software package SPSS for Windows v.15.0 (SPSS, Inc, Chicago, IL). Data from all groups are expressed as the mean +- SD. A probability value of less than 0.05 was accepted as statistically significant. Because the variances were not homogenous (Levene’s test statistic P b .05), post hoc Dunnett’s T3 analysis was performed to determine from which group any significant differences in the findings had arisen.

Results

The mean values for baseline (before the surgical procedure), before clamping and before sacrification (SAC) measurements as

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Table 1

Distribution of mean base, clamp, SAC, intact neurons, base progesterone, and after progesterone values of cases among groups

	Group 1		Group 2		Group 3		Toplam	P
	Mean +- SD		Mean +- SD		Mean +- SD		Mean +- SD
Base MAP	98.5 +- 7.74		79.83 +- 8.82		88.5 +- 9.77		88.94 +- 11.41	.008
Clamp MAP	92.67 +- 14.67		82.17 +- 5.12		80.83 +- 6.43		85.22 +- 10.62	.101
SAC MAP	82.5 +- 17.18		74.33 +- 18.79		55.67 +- 14.08		70.83 +- 19.56	.040
Base HR	288.17 +- 7.6		265.67 +- 22.92		262.83 +- 20.96		272.22 +- 20.9	.061
Clamp HR	239.67 +- 15.62		248.67 +- 36.52		264 +- 17.3		250.78 +- 25.67	.265
SAC HR	238 +- 14.14		240.5 +- 29.04		275.67 +- 34.09		251.39 +- 31.01	.052
Base SAT	99.17 +- 1.6		99.83 +- 0.41		99.67 +- 0.52		99.56 +- 0.98	.774
Clamp SAT	99 +- 1.67		99.17 +- 1.17		99.5 +- 0.55		99.22 +- 1.17	.438
SAC SAT	99.17 +- 0.75		99.17 +- 1.17		99.33 +- 0.82		99.22 +- 0.88	.001
Intact neuron	23.17 +- 4.49		37.17 +- 6.91		34.5 +- 4.59		31.61 +- 8.07	.217
Base PROG	0.72 +- 0.59		8.23 +- 14.58		0.24 +- 0.27		3.06 +- 8.77	.217
After PROG	0.74 +- 0.59		98.17 +- 142.95		117.51 +- 179.58		72.14 +- 135.13	.292

Values are given as mean +- SD; n = 6 in each group. Abbreviations: Sat: saturation; PROG, progesterone levels; clamp, clamping time.

well as the mean number of nonischemic neurons and mean progesterone levels for the 3 groups are listed in Table 1. Statistically significant differences were identified between the groups in their mean baseline mean arterial pressure (MAP), SAC MAP values (P b

.05). The analysis revealed that the average baseline MAP value for group 1 was significantly higher than that for group 2, and the mean SAC MAP value for group 1 was significantly higher than that for group 3 (P b .05). The number of intact neurons in group 1 was significantly lower than the number of intact neurons found in both groups 2 and 3 (P b .05). No other statistically significant differences were found between the groups, in terms of their mean progester- one heart rate (HR) and progesterone saturation (SAT) values (P N

.05) (Table 2). Although there seems to be a difference between the initial and follow-up progesterone measurements, the difference is not significantly different, as the confidence intervals cross zero (P N

.05) (Table 3). No statistically significant correlation was found between the number of live neurons in the ventral gray area and progesterone levels (either presurgery or postsurgery) in any of the groups (P N .05) (Table 4).

Discussion

Spinal cord ischemia is a severe pathologic condition, but the number of clinical studies on potential treatment options remains limited and insufficient. Unfortunately, many of the therapies that have been evaluated for their ability to treat spinal cord ischemia have failed to show much potential to yield effective improvements for patients. Consequently, there are no clear guidelines for the treatment of spinal cord ischemia. Animal studies have shown some benefit with certain agents, such as prostaglandins, nimodipine, naloxone, aden- osine, and magnesium, but there have been no prospective clinical studies evaluating their efficacy.

There have been case reports of the successful use of antithrom- botic therapy in spinal cord ischemia [13,14]. However, the practical utility of this method is limited because the efficacy antithrombotic

therapy is restricted to the first 3 to 4 hours after ischemia onset and because associated pathologies, such as Aorta dissection and vascular malformations, must be excluded before initiating treatment. Conse- quently, this method was shown to be appropriate for use in only 3% to 5% of patients. To the best of our knowledge, there is no literature on how much of the potentially infarcted area is salvaged by this method. Another potential treatment is Corticosteroid therapy [15]. There are numerous studies suggesting the neuroprotective effects of steroid hormone use after central nervous system injury [16,17]. Many studies have also focused on the potential benefit of glucocorticoids after spinal cord trauma [18-20]. The list of steroids that have neuroprotective effects has recently been lengthened. The neuroprotective effects of progesterones [7], androgens [21], and estrogens [22-24] after brain injuries have been reported. The neuroprotective effect of progesterone after spinal trauma has been demonstrated in rats, and its effect via binding to classical intracel- lular progesterone receptors (PRs) and to 25-Dx, a membrane- associated progesterone-binding protein, has been verified through immunocytochemical studies [25].

Recent studies have shown the neuroprotective effect of proges- terone in spinal cord and brain injuries, but its potential role in treating spinal cord ischemia, for which there currently exists no effective treatment protocol, had not been elucidated. Our study has demonstrated the neuroprotective effect of progesterone in spinal cord ischemia, which is associated with high Morbidity and mortality rates and demonstrates no clear clinical sign of onset, severely limiting the Clinical effectiveness of time-sensitive antithrombotic therapies. No other known treatments exist.

Our study has shown that progesterone administered immedi- ately after reperfusion, following a period of ischemia lasting up to 30 minutes, has a neuroprotective effect in spinal cord ischemia. In cases involving a longer period of ischemia and/or delayed treatment, however, it is difficult to estimate the Therapeutic effects of progesterone. Previous studies have demonstrated the neuroprotective effect of progesterone in traumatic brain injury up

Table 2

Distribution of mean progesterone values of the cases		among groups			Difference	SD	SEM	95% CI of the	t	df	P
Group 2	Group 3	Toplam	P		of means			difference

Table 3

The change between initial and later progesterone values of the cases in the groups

Mean +- SD			Mean +- SD		Mean +- SD							Lower	Upper
PROG MAP	54.83 +- 11.86	74.17 +- 14.7		64.5 +- 16.25		.031		Group 1	-0.03	0.06	0.02	-0.09	0.03	-1.12	5	.315
PROG HR	242.33 +- 35.38	269 +- 22.15		255.67 +- 31.4		.149		Group 2	-89.94	129.06	52.69	-225.38	45.50	-1.71	5	.149
PROG SAT	99.5 +- 0.84	99 +- 1.26		99.25 +- 1.06		.938		Group 3	-117.27	179.63	73.33	-305.79	71.24	-1.60	5	.171

Values are given as mean +- SD; n = 6 in each group. Abbreviation: PROG, progesterone treatment time.

Group 1 is the control group; groups 2 and 3, progesterone treatment group. Abbreviation: CI, confidence interval.

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Table 4

Correlation between number of intact neurons and base progesterone and after progesterone values of the cases

Group 1 Group 2 Group 3

	R	P		R	P		r	P
Base PROG	0.294	0.572	-0.062		0.907	-0.556		.252
After PROG	0.309	0.552	-0.348		0.499	0.472		.344

Base PROG, before progesterone treatment, progesterone levels in the blood. After PROG, after progesterone treatment progesterone levels in the blood.

to 24 hours after being administered. However, there are no studies currently evaluating potential treatments for spinal cord ischemia [9,10].

Conclusion

In the present study, we observed that progesterone has a neuroprotective effect in animal models of spinal cord ischemia when administered at the beginning of reperfusion following a 30- minute period of ischemia. We believe that the concomitant administration of progesterone with other protective therapies has the potential to reduce morbidity in cases of spinal cord ischemia.

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