Early Antiinflammatory Therapy Attenuates Brain Damage After Sah in Rats

Abstract Background Early inflammatory processes may play an important role in the development of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Experimental studies suggest that anti-inflammatory and membrane-stabilizing drugs might have beneficial effects, although the underlying mechanisms are not fully understood. The aim of this study was to investigate the effect of early treatment with methylprednisolone and minocycline on cerebral perfusion and EBI after experimental SAH. Methods Male Sprague-Dawley rats were subjected to SAH using the endovascular filament model. 30 minutes after SAH, they were randomly assigned to receive an intravenous injection of methylprednisolone (16mg/kg body weight, n=10), minocycline (45mg/kg body weight, n=10) or saline (n=11). Mean arterial blood pressure (MABP), intracranial pressure (ICP) and local cerebral blood flow (LCBF) over both hemispheres were recorded continuously for three hours following SAH. Neurological assessment was performed after 24 hours. Hippocampal damage was analyzed by immunohistochemical staining (caspase 3). Results Treatment with methylprednisolone or minocycline did not result in a significant improvement of MABP, ICP or LCBF. Animals of both treatment groups showed a non-significant trend to better neurological recovery compared to animals of the control group. Mortality was reduced and hippocampal damage significantly attenuated in both methylprednisolone and minocycline treated animals. Conclusion The results of this study suggest that inflammatory processes may play an important role in the pathophysiology of EBI after SAH. Early treatment with the anti-inflammatory drugs methylprednisolone or minocycline in the acute phase of SAH has the potential to reduce brain damage and exert a neuroprotective effect.


Introduction
The pathophysiology and treatment of early brain injury (EBI) have been more intensively focussed since drugs that successfully treated delayed cerebral vasospasm (DCV) failed to improve the neurological outcome of patients who suffered aneurysmal subarachnoid hemorrhage (SAH) [1,2]. The initial presentation, as documented by the Hunt/Hess and WFNS classifications, is still one of the most powerful predictors of long-term outcome. This further points out the importance of EBI [3,4].
Furthermore, it cannot be excluded that early pathophysiological changes may significantly influence mechanisms of delayed neurological deterioration occurring later in the course of the disease [5,6].
Apart from impaired cerebral perfusion, inflammatory pathways have been reported to contribute to EBI [7,8]. It is assumed that the accumulation of hemoglobin in the subarachnoid space and contact with collagen fibers trigger an immunological reaction [7], resulting in the activation of immunomodulators and, finally, diapedesis of leukocytes and macrophages into the brain parenchyma. Degranulation of macrophages, in turn, may result in an amplification of immunological cascades as well as the formation of reactive oxygen species, damaging neurons and glial cells and cerebral blood vessels and thus causing necrotic and apoptotic cell death [9].
Because of its effect on the glucocorticoid receptor, methylprednisolone (MTP) interferes with the production and effect of inflammatory cytokines, chemokines and cell-adhesion molecules [10]. In addition, glucocorticoids could also target pathomechanisms which inhibit the prostaglandin synthesis early after SAH and cause early vasospasm [11].
Recently Gomis et al. reported the results of a randomized controlled clinical trial, that patients who were treated with MTP after SAH showed significantly better clinical recovery one year after SAH [12].

Minocycline (MC) is a tetracycline antibiotic
with the potential to inhibit inflammatory pathways occurring early after SAH. A neuroprotective potential has been observed in neurodegenerative diseases and traumatic brain injury [13,14]. MC is a lipophilic drug and able to pass the blood brain barrier [15]. It exerts a number of anti-inflammatory mechanisms and may ameliorate the toxic effects of free subarachnoid hemoglobin, which is likely to be a trigger of inflammation after SAH [14,16].
It was the aim of this study to investigate the neuroprotective effects of MTP and MC on EBI after experimental SAH.

Material and Methods
For the experiments, 35 male Sprague-Dawley rats (purchased from Charles River, Sulzfeld, Germany) with a body weight between 270g and 370g were used. All experiments were approved by the regional authorities and the district government of Bavaria, Germany.

Anesthesia and monitoring
The rats were anesthetized with 4% Isoflurane

Intracranial pressure and local cerebral blood flow
To measure the intracranial pressure (ICP), a burr hole was drilled over the right frontal cortex 3 mm lateral and 0.5 mm anterior of the bregma.
The dura mater was opened diathermically. Two further burr holes were drilled 2 mm posterior and 5 mm lateral on each side of the bregma for the measurement of the local cerebral blood flow (LCBF) via laser-Doppler flowmetry (LDF).
Care was taken not to injure the dura mater. After all burr holes were completed, the animals were placed in a supine position with their head fixed in a stereotactic frame with non-rupture ear bars. A Camino ICP-probe (Integra Neurosciences, Plainsboro, NJ, USA) was advanced 2 mm into the brain using a micromanipulator. Using two further micromanipulators, rectangularly bent

Experimental groups and intervention
Four animals died or were sacrificed before the randomization process due to irregularities in the induction of anesthesia (n = 2) or endotracheal intubation (n = 2). The remaining 31 animals were randomly assigned to one of the following groups: stroke. [19]. In brief, the animal was placed into a large uncovered cage enriched with several stimuli (paper towels, wood tunnel, cardboard box and food pellets) together with a non-operated animal. After 10 minutes of acclimatization, its activity was evaluated after repeated manipulation (tail-holding, lateral push, repeated displacement of the animal).
Activity was graded following a 5-grade scale: 4) normal spontaneous activity, 3) slightly reduced spontaneous activity, 2) little or no spontaneous activity, but reaction on stimulus, 1) no activity on stimulus, 0) animal dead.
The examiner was blinded to the animal's treatment arm. Thereafter, the presence of hemiparesis was tested by assessment of the limb movement and forepaw stretching.

Quantification of subarachnoid blood and histological assessment
Following neurological assessment, the animals were again anesthetized with Isoflurane. Thereafter, an intraperitoneal injection of 1 ml of sodium pentobarbital (Narcoren, Boehringer Ingelheim, Germany) was administered and the animals were transcardially perfused with 4% paraformaldehyde. The animals' brains were removed and extent of SAH was quantified under the operation microscope using the Sugawara grading scale [20]. A score ranging from 0 to 18 was given based of the amount of blood cloths in six segments of the basal cistern.
After cryo-asservation the perfused brains

Statistical analysis
Statistical analysis was performed using

Physiological parameters
Values of arterial blood gases are depicted in Table 1. There were no significant differences between the groups regarding arterial pH, pO2 and pCO 2 throughout the experiment.
Mean arterial blood pressure, intracranial pressure and cerebral perfusion pressure

Discussion
In these experiments, a well established and standardized model of experimental SAH in rats was used to assess the neuroprotective potential of MTP and MC, two drugs that have long been approved by pharmacological agencies, are readily available and have a well known safety profile. The results suggest that early treatment with anti-inflammatory drugs can reduce early brain damage and improve mortality and neurological performance after The endovascular filament model is particularly useful to assess early pathophysiological changes and to compare them with parameters of early brain damage [22]. It was the particular aim of this work in the framework of a larger project to investigate whether there is a beneficial effect of treatment in the early phase of the disease. The substantial disadvantage of the endovascular perforation model is its high mortality as confirmed in the present experiments [17,23].
However, compared to other studies using this same model, pathophysiological changes were relatively mild in this series of animals [24].
In particular, ICP increased only moderately, the reason for which may be a relatively low   The pathophysiological cascade of early brain injury after SAH is complex offering a variety of possible targets for therapeutical intervention [28,29]. Extravasated hemoglobin, damaged endothelial barrier and free oxygen radicals lead to the formation of brain edema and activation of an inflammatory response that ultimately enhance or result in an uncontrolled apoptosis and/or necrosis [30,31]. In the last decade, the knowledge about the pathomechanisms of EBI has increased, but no early neuroprotective treatment after SAH has been introduced into clinical practice. In the present study, we investigated two anti-inflammatory drugs that have been in clinical use for many decades and are, thus ready for use.
MTP has been found to inhibit the induction of apoptosis, represses pro-inflammatory cells and mediators and the induction of suppressor cells [32]. In addition, it attenuates lipid peroxidation, reduces oxidative stress and stabilizes the endothelial barrier [33].
MTP has been used previously in the management of SAH in order to prevent or treat delayed cerebral vasospasm [34][35][36][37]. In  [13,14,35,46]. In addition, MC is a potent iron chelator forming iron complexes with the potential to attenuate the inflammatory response by the reduction of its neurotoxic stimulus [14].
In the present study, an improvement of CBF was not observed in the treatment groups.
In contrast, the treatment groups showed a tendency to lower CBF levels, suggesting that the improvement of neurological performance and attenuation of hippocampal damage are the result of intrinsic anti-inflammatory actions of those two drugs.
EBI after SAH seems to play a major role in the development of bad outcome in this patient population [49,50]. In a number of studies it has been shown that inflammatory pathways, oxidative damage caused by free oxygen radicals and disruption of the blood brain barrier lead to uncontrolled apoptosis and necrosis and, therefore, may result in neuronal damage [51,52]. and lipid peroxidation are possible mechanisms responsible for this finding [33,53].
MC also reduces hippocampal damage 24 hours after SAH, as well. The neuroprotective potential of the tetracycline antibiotic has been reported for a number of different neurological disorders [54]. The results of this study are in accordance with previous results obtained in experimental studies of SAH. Using a double injection model in rats, Guo and coworkers concluded that MC may also reduce EBI by inhibiting MMP-9 [43]. Furthermore, it may, in higher doses, reduce brain edema after SAH induced by the endovascular perforation model in rats [55]. Pathophysiological changes in rodent models may proceed more rapidly than in humans.
Therefore, the endpoint for neurological assessment and histological assessment was deliberately chosen.

Conclusion
In this experimental study, an attenuation of neuronal damage has been observed by therapy with the corticosteroid MTP and the tetracycline MC. Both substances have a number of potentially neuroprotective qualities. Since physiological parameters were not improved by treatment with either substance, we conclude that the strong antiinflammatory action is most likely to be the key factor for the beneficial effects.