Erythropoietin prevents delayed hemodynamic dysfunction after subarachnoid hemorrhage in a randomized controlled experimental setting

Abstract

Introduction

Erythropoietin (EPO) was proven as a promising approach for experimental subarachnoid hemorrhage (SAH). Clinical data are, however, inconclusive so far. A detailed characterization of specific EPO effects could facilitate the design of trials. The aim of the present investigation was, therefore, to characterize these effects on prevention of delayed proximal cerebral vasospasm (CVS), impaired microcirculation and cerebral blood flow (CBF) after experimental SAH.

Methods

27 male Sprague–Dawley rats were randomized in 3 groups: Sham, SAH control, and SAH EPO. SAH was induced by injection of 0.2 ml autologous blood into the cisterna magna on days 1 and 2. Animals of the SAH EPO group received 5000 iU rh EPO α 6 h after the 2nd SAH intravenously. Surviving animals were examined on day 5 by MR perfusion weighted imaging (PWI). Cerebral blood flow (CBF) and volume (CBV) were determined by PWI, proximal CVS by basilar artery (BA) diameter, and neuroprotection by hippocampal cell count (CA1–CA4).

Results

BA diameter was significantly reduced in both SAH groups, but improved significantly after EPO (Sham: 144 ± 3 μm, SAH control: 79 ± 6 μm, SAH EPO 109 ± 4 μm). The rrCBV ratio was 8.78 ± 0.72 Sham, 5.14 ± 1.73 SAH control, and 6.80 ± 0.44 SAH EPO. The improvement by EPO did not reach statistical significance. RrCBF ratio was also significantly reduced in both SAH groups, but was significantly improved by EPO (Sham: 8.78 ± 0.34, SAH control: 4.26 ± 1.05, SAH EPO 5.85 ± 0.46). Surviving neuronal cells were significantly reduced in SAH controls in all areas, but in SAH EPO only in CA1.

Conclusion

The present data suggest that an EPO application in a timely distance to the SAH is sufficient to prevent delayed proximal CVS, but that the doses were insufficient to improve microcirculation or to be directly neuroprotective.

Introduction

The growth factor erythropoietin (EPO) is a protein belonging to the type I cytokine superfamily and is mainly known as potent stimulator of erythropoiesis [1], [2], [3] or as a drug for doping in sports [4]. During the last decade there was, however, growing evidence for several tissue protective properties and effects which reduce the need of blood transfusion or which stabilize the circulation in critical care patients [1], [2], [5]. Furthermore, it was proven in a large clinical trial that the overall survival rate improved in critical ill patients treated with a weekly application of EPO [6]. Therefore, EPO came into the center of interest for the treatment of intensive care patients in several medical disciplines, among them neurosciences [2], [3], [7], [8], [9]. Beginning with animal models, the neuroprotective effects of EPO were characterized to prevent brain damage under several circumstances most notably for focal cerebral ischemia [9], [10], traumatic brain injury [1], [11], [12], and subarachnoid hemorrhage [13], [14], [15], [16], [17], [18].

Consequently, clinical trials were performed to translate such promising results from bench to bedside. Regarding traumatic brain injury, data from a case control study are available suggesting a significant reduction of mortality by an EPO analogon [19]. Currently some clinical trials are conducted and, therefore, prospective randomized data may be available in the near future [1]. For the treatment of ischemic stroke, a Phase III study was conducted [20] after promising results of a pilot study [21]. In this Phase III study, however, human recombinant (hr) EPO failed to improve neurologic outcome or infarct size [20]. In contrast mortality rate was even enhanced in the EPO group, particularly in the subgroup in which EPO was used in combination with recombinant tissue plasminogen activator (rTPA). Adverse events like intracerebral hemorrhage, brain edema and thrombosis were also enhanced in the EPO group [20]. Similarly, the rate of thrombosis was also increased in the larger clinical trials mentioned above [6], [19]. Therefore, these disappointing results raised not only the question for the efficacy of EPO for neuroprotection, but also for the safety of its application. A direct consequence of this observation was the truncation of a clinical trial in patients with aneurismal SAH (NCT00626574). So far data from 2 randomized clinical trials including 73 and 80 patients are available for the treatment of SAH with EPO [22], [23]. These two trials provide, however, somewhat discrepant results, even though dose and subtype of EPO were comparable. The first trial was truncated after 73 patients and no significant improvement by hr EPO α could be proven [23]. In the second trial a significant reduction of delayed cerebral vasospasm (CVS) could be observed, which was, however, only determined by transcranial Doppler (TCD). Regarding the clinical outcome, there was a trend for an improvement at discharge, which could not be reproduced in the 6 month follow-up [22]. The available data for the treatment of aneurismal SAH with EPO are, therefore, not conclusive at present.

Several different pathophysiological mechanisms are adding to a poor outcome after aneurismal SAH which occurs in a more or less defined time course [24], [25], [26]. In the acute phase the rupture of the aneurysm results in a rapid increase of the intracranial pressure, which reduces the cerebral blood flow (CBF), and may lead to cerebral ischemia [24], [25]. This ischemia could result in tissue hypoxia and brain edema [25], and may initiate a cascade of several mechanisms inside the brain tissue [27] and cerebrovasculature [26]. These initial effects of SAH were termed early brain injury (EBI) and are mainly dealing with the effects during the first 72 h after SAH [27]. This EBI can, therefore, be discriminated from delayed ischemic neurological deficits (DIND), which occur after this initial phase [24], [26]. Pathophysiological background of this DIND is presumably the subarachnoid blood and its degradation products, which are affecting the cerebrovasculature and/or the brain surface. These effects lead to an impaired CBF by narrowing of the proximal cerebral arteries, which represents the “classic” CVS and/or a disturbed microcirculation [26], [28]. This reduced CBF results in cerebral ischemia or (depending on the extent) to cerebral infarction in about 15% of the cases [24], [25], [26], [29], [30]. Even for patients treated in specialized centers, the rate of poor outcome after SAH remains to be more than 30% and mortality up to 10% [24], [29], [30]. The initial bleeding, a re-bleeding before aneurysm treatment, and treatment associated complications can be considered as reasons for a poor outcome in about 60%. DIND and intensive care treatment associated reasons seem to be responsible for the remaining 40% of the cases [24], [25], [26]. Several potential and promising targets for a prophylaxis or a treatment with EPO after SAH are, therefore, remaining, even in the knowledge of the disappointing results of the stroke trial [20]. To avoid further discouraging results from clinical trials, experimental investigations could, however, be beneficial to define the optimal time, dose and subtype of EPO [31], [32]. Furthermore, the target of an EPO treatment beneath the different pathophysiological mechanisms after SAH should be defined as exactly as possible to balance its beneficial against its potential side effects [6], [20]. Such data may help to find e.g. indication criteria for EPO treatment of DIND after SAH. This may facilitate to develop improved protocols for clinical trials which may have a chance to reveal a significant effect.

Accordingly, the aim of the present investigation was to characterize the effect of a single hr EPO alpha application on the prevention of delayed CBF impairment by proximal CVS or disturbed microcirculation in an animal SAH model focussed on these delayed mechanisms.