Heparin Reduces Neuroinflammation and Transsynaptic Neuronal Apoptosis in a Model of Subarachnoid Hemorrhage
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Subarachnoid hemorrhage (SAH) can lead to disabling motor, cognitive, and neuropsychological abnormalities. Part of the secondary injury to cerebral tissues associated with SAH is attributable to the neuroinflammatory response induced by blood. Heparin is a pleiotropic compound that reduces inflammatory responses in conditions outside the central nervous system. Using a model of SAH devoid of global insult, we evaluated the effect of delayed intravenous (IV) infusion of heparin, at a dose that does not produce therapeutic anticoagulation, on neuroinflammation, myelin preservation, and apoptosis. Adult male rats underwent bilateral stereotactic injections of autologous blood (50 μL) into the subarachnoid space of the entorhinal cortex. The rats were implanted with mini-osmotic pumps that delivered either vehicle or unfractionated heparin (10 U/kg/h IV) beginning 12 h after SAH. No mechanical or hemorrhagic injury was observed in the hippocampus. In vehicle controls assessed at 48 h, SAH was associated with robust neuroinflammation in the adjacent cortex [neutrophils, activated phagocytic microglia, nuclear factor-kappa B, tumor necrosis factor-alpha, and interleukin-1beta] and neurodegeneration (Fluoro-Jade C staining and loss of NeuN). In the hippocampus, a muted neuroinflammatory response was indicated by Iba1-positive, ED1-negative microglia exhibiting an activated morphology. The perforant pathway showed Fluoro-Jade C staining and demyelination, and granule cells of the dentate gyrus had pyknotic nuclei, labeled with Fluoro-Jade C and showed upregulation of cleaved caspase-3, consistent with transsynaptic apoptosis. Administration of heparin significantly reduced neuroinflammation, demyelination, and transsynaptic apoptosis. We conclude that delayed IV infusion of low-dose unfractionated heparin may attenuate adverse neuroinflammatory effects of SAH.
KeywordsSubarachnoid hemorrhage Neuroinflammation Microglia Heparin Transsynaptic apoptosis Rat
Subarachnoid hemorrhage (SAH) comprises 5–7 % of all strokes [30, 38]. Acutely, 30 % or more of patients die from the initial hemorrhage or rehemorrhage [30, 38]. Patients who survive are at high risk for potentially severe neurological and neuropsychological complications that develop during the subsequent days and weeks, with well over half succumbing to cerebral vasospasm, stroke, hydrocephalus, or cognitive and neuropsychological deficits. Among the worst cases are those in which the hemorrhage involves the eloquent cortex, a situation frequently encountered with rupture of middle cerebral artery aneurysms [55, 56]. However, even among patients with mild hemorrhages (World Federation of Neurosurgical Societies grade 1) who do not suffer clinical or radiological vasospasm and who do not experience perioperative complications, long-term psychosocial and cognitive difficulties are still very common [6, 10].
Collectively, the various neurological and neuropsychological abnormalities brought on by SAH may be referred to as “delayed neurological deficits” (DNDs) . Historically, the most devastating cause of DNDs was considered to be vasospasm leading to ischemia and stroke. Recently, a large clinical trial demonstrated that successful prevention of angiographic vasospasm by inhibition of endothelin signaling does not translate into improved clinical outcomes . This landmark finding indicates that other mechanisms of secondary injury induced by blood, possibly unrelated to ischemia, play a crucial role in the development of DNDs [5, 8, 21, 31].
SAH has long been known to induce an inflammatory response in blood vessels and neuronal tissues [11, 17, 24, 29, 52, 61, 64, 72, 73, 77, 78, 84]. Blood incites a robust inflammatory response in the central nervous system that is attributable, in part, to oxidative stress . Many conventional markers of inflammation are activated or upregulated by SAH, including leukocytes, microglia, immune complexes, complement, C-reactive protein, and numerous cytokines and chemokines [11, 22, 24, 37, 62, 73, 77]. Despite convincing evidence for the involvement of inflammation, block of conventional inflammatory/immune pathways using a variety of agents (glucocorticoids, lazaroids, iron chelators, cyclosporine, tacrolimus, and nonsteroidal anti-inflammatory drugs) has been of limited benefit in ameliorating DNDs associated with SAH, both in animal models and in humans [11, 72]. Nevertheless, recent studies examining nonconventional anti-inflammatory agents have shown promise [73, 77].
Heparin is a member of a family of polyanionic polysaccharides called glycosaminoglycans, composed of hexuronic acid and d-glucosamine residues joined by glycosidic linkages . Although clinically utilized almost exclusively as an anticoagulant, heparin binding can interrupt numerous biological pathways . Heparin is a pleiotropic drug that has long been recognized to have broad anti-inflammatory and immunomodulatory activities that are independent of its anticoagulant effect [18, 44, 45, 65, 80, 86]. On the extensive list of heparin-binding proteins are several important cytokines and all chemokines [18, 80, 86].
We recently reviewed the potential benefits of heparin as a pleiotropic, prophylactic agent in the context of SAH, showing how heparin can potentially antagonize most of the pathophysiological mechanisms known to be activated following SAH . Here, we evaluated the effect of delayed intravenous (IV) infusion of heparin, at a dose that does not produce therapeutic anticoagulation [4, 42], in a model of SAH involving the entorhinal cortex, the part of the temporal lobe that projects directly to the hippocampus via the perforant pathway. In this model, SAH was associated with robust neuroinflammation in the adjacent cortex and white matter, with muted neuroinflammation in the hippocampus, with demyelination of the perforant pathway, and with transsynaptic apoptosis in the dentate gyrus. Here, we report that delayed IV infusion of low-dose heparin significantly reduced neuroinflammation, demyelination, and transsynaptic apoptosis.
Subjects and Experimental Series
All experimental procedures were approved by the University of Maryland Institutional Animal Care and Use Committee. Male Wistar rats (300–350 g; Harlan, Indianapolis, IN, USA) were used in this study, which was comprised of two experimental series: In series 1, eight rats underwent bilateral SAH (see below), three rats underwent unilateral SAH (right side only), and three rats underwent sham injury [same procedure but with injection of normal saline (NS) instead of blood]. They were euthanized at 48 h. These brains were used for pathological and histochemical characterization of the model, including assessment for infarction using 2,3,5-triphenyltetrazolium chloride (TTC) staining (three rats with bilateral SAH), assessment of tissue damage (Fluoro-Jade C staining) with saline vs. blood (three rats each with unilateral saline vs. blood), and evaluation of the hemorrhage location with hematoxylin and eosin (H&E) staining (five rats with bilateral SAH). In series 2, 16 rats underwent bilateral SAH and were implanted with mini-osmotic pumps that delivered either NS (8 rats) or heparin (8 rats; see below). These brains were used for further histochemical and immunohistochemical characterization of the model and to assess the effect of heparin treatment. In accordance with “good laboratory practice,” surgical procedures were conducted by investigators blinded to treatment.
Rat Model of SAH
Anesthetized (ketamine 60 mg/kg and xylazine 7.5 mg/kg intraperitoneally) rats spontaneously breathed room air supplemented with oxygen to maintain 90 % < sO2 < 98 % by pulse oximetry. Temperature was maintained at 37°C using a heating pad regulated by a rectal temperature sensor (Harvard Apparatus, Holliston, MA, USA). Surgical procedures were carried out using aseptic techniques.
SAH was produced by injecting 50 μL of autologous blood under stereotactic guidance into the subarachnoid space of the entorhinal cortex, bilaterally. First, the tail artery was cannulated (PE-20 tubing) under microscopic visualization. The head was fixed in a stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA) and a midline scalp incision was made. One burr hole was placed at (bregma and midline as reference points) anterior–posterior, −7.7 mm; lateral, +5 mm; a second burr hole was placed at anterior–posterior, −7.7 mm; lateral, −5 mm. The dura at each burr hole was pierced sharply. Then, 600 μL of blood was aspirated from the tail artery; the last 125 μL of nonheparinized blood was aspirated into a syringe equipped with a 22-mm-long blunt, tapered-tip needle (Hamilton custom tapered syringe, 7732-01; Hamilton Co., Reno, NV, USA). The syringe was mounted in the stereotaxic apparatus and the tip coordinates were registered. Beginning at the burr hole on the right side, the needle was advanced to the z-coordinate of −7.0, then was advanced very slowly until the curvature of the posterior aspect of the middle fossa was reached, as indicated by the needle shifting slightly anteriorly [at the z-coordinate (mean ± SD), −8.3 ± 0.4 mm; range, −7.3 to −9.2 mm]. Use of a blunt, tapered-tip needle prevented the dura and bone from being pierced, allowing reliable access to the subarachnoid space. Without adjusting the depth, 50 μL of blood was injected over 5 min, after which 5 min was allowed to elapse before slowly withdrawing the needle. The procedure was repeated on the left side.
In rats that would be administered drug, IV access to the external jugular vein was obtained under microscopic visualization. A mini-osmotic pump (1.0 μL/h; Azlet 2001, Durect Corporation, Cupertino, CA, USA) fitted with a rat jugular vein catheter (#0007710, Alzet, Durect Corporation, Cupertino, CA, USA) was implanted. The catheter was filled sequentially with 11 μL of NS, 1 μL of air, and the remainder with the solution to be administered; the first 12 μL in the catheter provided a 12-h delay in the start of drug administration . The pumps were filled either with NS or with heparin sodium (5,000 USP U/ml; APP Pharmaceuticals, LLC, Schaumburg, IL, USA) in NS (1 ml of heparin stock plus 0.43 ml NS) that delivered 10 U/kg/h. Therapeutic anticoagulation in the rat requires constant infusion of 75 U/kg/h of heparin .
After transcardiac perfusion/fixation with 10 % neutral buffered formalin, the brains were cryoprotected with 30 % sucrose. For routine histology, cryosections (10 μm) were stained with H&E and examined using light microscopy.
To assess myelin, cryosections (10 μm) were stained with Black Gold II according to manufacturer’s protocol (Black Gold II Myelin Staining Kit, AG105, Millipore, Temecula, CA, USA). Briefly, slides were first rehydrated and incubated in prewarmed 0.3 % Black Gold II solution at 60°C for 15 min. Sections were then rinsed twice, and then incubated in 1 % sodium thiosulfate solution for 3 min at 60°C. After several rinses with Milli-Q water, the sections were counterstained with cresyl violet to visualize nuclei, were coverslipped, and were examined using light microscopy.
To identify degenerating neurons and white matter tracts, cryosections (10 μm) were labeled with Fluoro-Jade C histofluorescent stain  according to the manufacturer’s protocol (Histo-Chem, Jefferson, AR, USA). Sections were coverslipped with nonpolar mounting medium (Cytoseal XYL, Richard-Allan Scientific, Kalamazoo, MI, USA) and were examined with an epifluorescence microscope (Nikon Eclipse 90i) under a fluorescein isothiocyanate filter, with care being taken to limit the time of exposure in order to reduce photobleaching.
Cryosections (10 μm) were first blocked (5 % goat serum, Sigma + 0.2 % Triton X-100 for 1 h at room temperature) and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (1:1,000; Wako Chemicals, Richmond, VA, USA); mouse anti-ED-1 (1:200; MAB1435; Millipore, Temecula, CA, USA); rabbit antimyeloperoxidase (1:200; A0398; Dako, Carpinteria, CA, USA); rabbit antinuclear factor-kappa B (p65) (1:200; sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anticleaved caspase-3 (1:200; Asp175; Cell Signaling Technologies, Beverly, MA, USA); mouse antiglial fibrillary acidic protein (GFAP) (1:500; CY3 conjugated; C-9205; Sigma, St. Louis, MO, USA); goat antitumor necrosis factor-alpha (TNFα) (1:100; N-19, #SC-1350; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-interleukin-1beta (IL-1β; #AB9787; Abcam, Cambridge MA, USA).
After several rinses in phosphate-buffered saline, the slides were incubated for 1 h with fluorescent-labeled species-appropriate secondary antibodies (1:500; Alexa Flour 488 and Alexa Flour 555; Invitrogen, Molecular Probes, Eugene, OR, USA) at room temperature. Omission of primary antibody was used as a negative control. The sections were coverslipped with polar mounting medium containing antifade reagent and 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Eugene, OR, USA) and were examined using epifluorescence microscopy (Nikon Eclipse 90i; Nikon Instruments Inc., Melville, NY, USA). In some cases, for the sake of clarity of presentation, epifluorescence images are presented in the figures as “inverse” images, wherein the brightness value of each pixel is converted to the inverse value on a 256-step scale.
Quantifications comparing the effect of vehicle vs. heparin were made using the right side of the brain. Unbiased measurements of specific labeling within regions of interest (ROI) were obtained using NIS-Elements AR software (Nikon Instruments, Melville, NY, USA) from sections immunolabeled in a single batch, as previously described [26, 60]. All ROI images for a given signal were captured using uniform parameters of magnification, area, exposure, and gain. The following ROIs were defined: (1) for MPO, ED1, p65, TNFα, and IL-1β in the entorhinal cortex, a rectangular ROI, 1 × 1 mm, that was positioned on the pial surface; (2) for Iba1 in the hippocampus, a rectangular ROI that was positioned on the perforant pathway (see Fig. 6); (3) for Black Gold II in the hippocampus, a rectangular ROI that was positioned in the region between the perforant pathway and the dentate gyrus (see Fig. 7); (4) for Fluoro-Jade C in the hippocampus, a rectangular ROI, 450 × 360 μm, that was positioned at the hilus of the dentate gyrus. Segmentation analysis was performed by computing a histogram of pixel intensity for a particular ROI. Specific labeling was defined as pixels with signal intensity greater than two times that of background. For MPO, ED1, and p65, we performed object counts for specific labeling, which for MPO and ED1 corresponded to the number of cells. For Iba1 and Black Gold II, the area occupied by pixels with specific labeling was used to determine the percent area with specific labeling (%ROI). For Fluoro-Jade C, we counted the number of neurons within the ROI.
Calculations were performed with OriginPro8 (OriginLab Corp., Northampton, MA, USA). Statistical comparisons were made using Student’s t test or a one-way analysis of variance with Fisher’s post hoc comparison, as appropriate.
SAH Involving the Entorhinal Cortex
Neurodegeneration in the Entorhinal Cortex
Neuroinflammation in the Entorhinal Cortex
Effect of Heparin on Neuroinflammation in the Entorhinal Cortex
Effect of Heparin on Neuroinflammation in the Hippocampus
We studied neuroinflammation in a model of SAH that involved the eloquent cortex and that was devoid of a global insult. To characterize microglial activation, we used two antibodies that convey complementary information. The ED1 antibody is a macrophage marker that recognizes an intracytoplasmic, lysosomal antigen whose expression increases during phagocytic activity in monocytes and other tissue macrophages, including microglia [3, 15, 28]. The Iba1 antibody is a marker directed against a microglia-specific antigen that identifies nonactivated (resting), as well as activated but nonphagocytic microglia that are ED1-negative . Using these and other antibodies, we found a neuroinflammatory response in the entorhinal cortex adjacent to the hemorrhage that was robust, with invasion of neutrophils, ED1-positive cells, phagocytic microglia, and macrophages, and prominent labeling for NF-κB, TNFα, and IL-1β [16, 77]. This robust neuroinflammatory response was associated with significant neurodegeneration, marked by Fluoro-Jade C staining and loss of NeuN labeling. The robust inflammatory response in the entorhinal cortex was very different from the muted inflammatory response observed remotely in the hippocampus, where there was no labeling for MPO, ED1, or NF-κB and where the principal evidence for neuroinflammation was the appearance of Iba1-positive microglia that exhibited an activated morphology. Overall, our findings on neuroinflammation associated with SAH are consistent with previous reports [2, 11, 17, 24, 29, 52, 61, 64, 72, 73, 77, 78, 84]. What may be an importance difference, however, is that, in many of the previous reports, either the model used (endovascular puncture) or the amount of blood injected (200–500 μL) could potentially have been associated with a widespread or global ischemic insult, whereas in our experiments, the manner of injection and the amount of blood injected (50 μL on each side) were highly unlikely to produce an ischemic injury, as confirmed by the lack of vasoconstriction or vasospasm of vessels in the subarachnoid space and by uniformly positive tissue staining with TTC.
The major finding of the present study is that delayed IV infusion of heparin, at a dose that does not produce therapeutic anticoagulation, significantly reduced neuroinflammation associated with SAH, both the proximate effect in the entorhinal cortex, as well as the remote effect in the hippocampus. Heparin is a pleiotropic drug that has long been recognized to have broad anti-inflammatory and immunomodulatory activities that are independent of its anticoagulant effect [18, 44, 45, 65, 80, 86]. More than 100 heparin-binding proteins are known , with the growing list including numerous plasma proteins, proteins released from platelets, cytokines, chemokines, and other small, biologically active molecules, as well as endothelial cells themselves [14, 25, 32, 36, 51, 65]. The anti-inflammatory effects of heparin are due to a number of interactions: (1) inhibition of complement activation [23, 83]; (2) binding to the leukocyte adhesion molecules, P- and L-selectin [65, 81]; (3) inhibition of the cationic neutrophil proteases, human leukocyte elastase, and cathepsin G [23, 65]; (4) disruption of Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) ; (5) inhibition of RAGE ligation by its many proinflammatory ligands, including the advanced glycation end product, carboxymethyl lysine–bovine serum albumin, the nuclear protein, high mobility group box protein-1, and S100 calgranulins [46, 47, 53, 65, 67]; (6) inhibition of the nuclear translocation of NF-κB and a decrease in NF-κB–DNA binding in the endothelium .
A recent comprehensive review showed that heparin exhibits a broad diversity of biological effects, many of which can be directly linked to pathophysiological mechanisms that have been implicated in SAH-induced DNDs . Among its wide-ranging effects, heparin (1) complexes with free hemoglobin, including oxyhemoglobin ; (2) blocks the activity of free radicals including reactive oxygen species ; (3) antagonizes endothelin-mediated vasoconstriction [12, 41, 85]; (4) accelerates antithrombin-III-mediated degradation of thrombin  which is implicated in protracted vasoconstriction ; (5) binds to several growth factors, thereby imparting antimitogenic [33, 34, 39] and antifibrotic  effects; and as reviewed above, (6) exerts potent multitargeted anti-inflammatory effects. The efficacy reported here for heparin against neuroinflammation, combined with the fact that the dose we used was less than that required for therapeutic anticoagulation [4, 42], suggests that heparin may be a safe prophylactic agent for reducing DNDs associated with SAH.
Despite the absence of a global insult, we identified extensive cell degeneration and cell death in this model, especially in the entorhinal cortex and white matter near the SAH. Hippocampal and cortical cell death and neuronal apoptosis have been previously documented, both in models of SAH and in humans with SAH [7, 54], but the trigger for apoptosis is not always known. With the endovascular perforation models, cell death has been attributed to a global insult, e.g., an elevation of the intracranial pressure resulting in global ischemia/hypoxia [19, 58, 59, 63]. However, several studies have shown that SAH produced by cautious blood injection can be also associated with neuronal apoptosis, independent of increased intracranial pressure [49, 50, 68, 82]. In the absence of a global ischemic insult, the actual trigger for cell death is often uncertain .
Given the absence of a global insult, the absence of direct mechanical or hemorrhagic injury to the hippocampus, and the muted inflammatory response in the hippocampus, we were initially surprised to find apoptosis of granule cells of the dentate gyrus. Apoptosis of granule cells was characterized by pyknotic nuclei that stained with Fluoro-Jade and showed cleaved caspase-3. The fact that we were injuring the entorhinal cortex gave rise to the possibility that, if not attributable to a global insult or to direct injury, apoptosis might be due instead to a transsynaptic process. Transsynaptic degeneration and transsynaptic apoptosis have been well documented under a variety of conditions in many brain regions following deafferentation, including the system of entorhinal cortex–perforant pathway–dentate gyrus that we studied here [27, 40, 71]. Our findings of degeneration/demyelination of the perforant pathway, as indicated by Fluoro-Jade C staining , and diminished labeling with Black Gold II are consistent with deafferentation of dentate gyrus cells secondary to the robust inflammatory response in the entorhinal cortex. Injury to the perforant pathway that causes deafferentation of granule cells renders these cells susceptible to excitotoxic injury and apoptotic death . Moreover, transsynaptic apoptosis is worsened by neuroinflammation , as we observed here. Importantly, demyelination and transsynaptic apoptosis in our model were largely blocked by delayed IV infusion of low-dose heparin.
To our knowledge, transsynaptic apoptosis has not been previously identified in SAH. The potential implications of transsynaptic apoptosis in SAH may be large, especially if SAH involves the eloquent cortex. Distinct from generally recognized triggers of apoptosis, such as direct tissue trauma, ischemia, inflammation, etc., transsynaptic apoptosis involves the spread of cell death beyond the boundaries of the immediate injury to involve otherwise uninjured neurons that are an axon’s length away. This phenomenon may help to explain the prevalence of cognitive and neuropsychological deficits that commonly afflict humans with “mild-to-moderate” SAH who do not suffer a global insult [6, 10].
In summary, our findings here support previous work showing a robust neuroinflammatory response in the cortex underlying SAH which can lead to nearby tissue damage and cell death, as well as remote transsynaptic apoptosis. Our studies also show that delayed IV infusion of low-dose heparin exerts a powerful anti-inflammatory effect that lessens tissue injury and reduces transsynaptic apoptosis.
This work was supported by grants to JMS from the National Institute of Neurological Disorders and Stroke (NS061808) and the National Heart, Lung and Blood Institute (HL082517).
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- 21.Etminan N, Vergouwen MD, Ilodigwe D, et al. Effect of pharmaceutical treatment on vasospasm, delayed cerebral ischemia, and clinical outcome in patients with aneurysmal subarachnoid hemorrhage: a systematic review and meta-analysis. J Cereb Blood Flow Metab. 2011;31:1443–51.PubMedCrossRefGoogle Scholar
- 48.Macdonald RL, Higashida RT, Keller E, et al. Clazosentan, an endothelin receptor antagonist, in patients with aneurysmal subarachnoid haemorrhage undergoing surgical clipping: a randomised, double-blind, placebo-controlled phase 3 trial (CONSCIOUS-2). Lancet Neurol. 2011;10:618–25.PubMedCrossRefGoogle Scholar