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Intrathecal application with liposome-entrapped Fasudil for cerebral vasospasm followingsubarachnoid hemorrhage in rats
Journal of Clinical Neuroscience (2001) 8(6), 557±561 & 2001 Harcourt Publishers Ltd DOI: 10.1054/jocn.2001.0998, available online at http://www.idealibrary.com on
Laboratory studies
Intrathecal application with liposome-entrapped Fasudil for cerebral vasospasm following subarachnoid hemorrhage in rats Yoshihiro Takanashi1 MD PHD, Tatsuhiro Ishida2 PHD, Toshinari Meguro3 MD, Marc J. Kirchmeier2 PHD, Theresa M. Allen2 PHD, John H. Zhang3 MD PHD 1
Department of Neurosurgery, Yokohama City University, Yokohama, Japan, 2Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, 3Department of Neurosurgery, University of Mississippi Medical Center, Jackson, Mississippi, USA
Summary To date, the pharmacological approach to cerebral vasospasm following subarachnoid hemorrhage has been hampered in part by an inability to attain sufficiently high concentrations of vasodilator drugs in the cerebrospinal fluid (CSF). To overcome this limitation of current drug therapy, we have developed a sustained-release preparation of protein kinase inhibitor Fasudil. Cerebral vasospasm in rats was induced by double-injection method. Treated rats received 0.417 mg liposome-entrapped Fasudil via the cisterna magna and control rats received drug-free liposomes in the same manner. The diameter of the basilar artery was assessed at 7 days after the initial blood injection. Vasoconstriction of the rat basilar artery was significantly reduced in group treated with liposomal Fasudil compared to the control group (treated group: 87.7 6.18%, n 10; control group: 66.3 9.82%, n 10; ***P < 0.001). This new approach for cerebral vasospasm may have significant potential for use in the clinical setting. & 2001 Harcourt Publishers Ltd Keywords: cerebral vasospasm, drug delivery systems, Fasudil, liposomes, rat, subarachnoid hemorrhage
INTRODUCTION Cerebral arterial vasospasm with delayed ischemic neurological deficit occurs in one-third of patients with aneurysmal subarachnoid hemorrhage (SAH).1 Despite considerable advances in perioperative management of these patients, including the use of calcium antagonists, cerebral vasospasm, which is the delayed narrowing of major arteries at the base of the brain, still remains a major cause of morbidity and mortality after SAH.2 Recently, Fasudil hydrochloride (1-5-isoquinolinesulphonylhomopiperazine), which has a vasodilating effect, has been used in patients with SAH and encouraging data have been reported for prevention of cerebral vasospasm.3,4 Experimental work has repeatedly shown that vasodilator drugs can reverse established angiographically identified vasospasm when administered by the intrathecal route, despite being ineffective when administered by the intravenous or intra-arterial route.5,6 Although intrathecal vasodilatory therapy for the treatment of cerebral vasospasm could minimise the risk of systemic adverse effects such as hypotension, it is difficult to continuously maintain a therapeutic drug concentration in the CSF. The limited application of intrathecal route, especially in clinical studies, is associated with the troublesome procedure and sideeffects such as meningitis as the result of long-term indwelling catheters. Moreover, the time during which drug concentrations remain in the therapeutic window may be transient even if bolus application by the intrathecal route was feasible. We have devised a novel method for the prevention of experimental vasospasm by which a sustained-release form of the drug can maintain a therapeutic concentration in the CSF within Received 28 August 2000 Accepted 31 October 2000 Correspondence to: Yoshihiro Takanashi MD, PhD, Department of Neurosurgery, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Japan, 236-0004. Tel.: 81 45 787-2663; Fax: 81 45 783 6121; E-mail: [email protected]
a therapeutic window. The aim of the present study is to evaluate whether intrathecal administration of the liposomal Fasudil can prevent cerebral vasospasm following SAH in a rat model. MATERIALS AND METHODS Preparation of liposomes Liposomes were made with two different lipid compositions. Classical liposomes were composed of hydrogenated soy phosphatidylcholine : cholesterol (HSPC : CHOL) at 2 : 1 molar ratio. Stealth liposomes were composed of HSPC : CHOL : methoxypolyethyleneglycol (Mr 2000) covalently linked to distearoylphosphatidylethanolamine (mPEG2000-DSPE) at 2 : 1 : 0.1 molar ratio. For Fasudil-loaded liposomes, we used remote loading with an ammonium sulfate gradient to encapsulate the drug.7 Dried lipid films were hydrated in 250 mM ammonium sulfate (pH 3.0). Then, extrusion (Lipex Biomembranes Extruder, Vancouver, British Columbia, Canada) was sequentially carried out to produce homogeneously sized liposome preparations, using a series of polycarbonate filters (Nuclepore Corp., Pleasanton, CA, USA) with pore sizes ranging from 0.4 to 0.08 mm. The mean diameter of liposomes which was determined by dynamic light scattering using a Brookhaven B190 submicron particle size analyzer (Brookhaven Instruments Corp., Holtsville, NY, USA) was in the range of 110 10 nm. The external buffer was then exchanged by eluting through a Sephadex G50 column equilibrated with 10% sucrose (pH 8.0). Fasudil (Sigma Chemical Co., St Louis, MO, USA) was loaded into the liposomes at a phospholipid (PL): Fasudil ratio of 1 : 0.4 (w/w) and incubated for 1 h at 65 C. Liposome-entrapped Fasudil was separated from free Fasudil using a Sephadex G50 column eluted with 25 mM N-2-hydroxyethyl piperazine-N 0 -2-ethansulfonic acid (HEPES) and 140 mM NaCl buffer (pH 7.4). Spectrophotometry ( 320 nm) finally determined the concentration of the liposome-entrapped Fasudil, and PL concentrations were determined by means of the Bartlett colorimetric assay.8 The loading efficiency of Fasudil into liposomes was also determined. 557
558 Takanashi et al.
Releasing property of liposome-entrapped Fasudil in vitro The in vitro drug-release kinetics of the liposome-entrapped Fasudil was measured in control CSF (Sigma Chemical Co, St Louis, MO, USA). Either classical liposomes or Stealth liposomes (0.8 ml) containing 0.417 mg of Fasudil and 1.303 mmol PL were incubated in 4.2 ml of CSF at 37 C for 7 days. Periodically, 0.5 ml of the medium was withdrawn and replaced with an equivalent volume of fresh CSF to mimic CSF turnover in vivo. Free Fasudil was separated from liposome-entrapped Fasudil using a Sephadex CL-4B column equilbrated with HEPES buffer (pH 7.4), as above. Following dissolution in 100% methanol, the concentration of the Fasudil in liposomes was then determined by spectrophotometry (=320 nm), and PL concentrations were determined using the Bartlett colorimetric assay.8 Experimental model of SAH and study design Male Sprague±Dawley rats weighing 350±400 g were used for the experiments. Animals were housed in the vivarium with free access to water and food in a 12-h, day±night cycle. Approval from the institutional ethics committee (University of Mississippi Medical Center) was obtained for all procedures. SAH in a rat was induced by injection of autologous arterial blood into the cisterna magna twice.9,10 Rats were anethsthetised by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and allowed to breathe spontaneously. Anesthesia was maintained by additional injections of ketamine and xylazine as needed. Rectal temperature was maintained at approximately 37 0.5 C throughout the surgical procedure using a heating pad and overhead lamp. Under sterile condition, rats were placed in a head flexion angle of 30 with a rolled-up gauze under the neck. The atlanto-occipital membrane was fully exposed through a midline skin incision while dissecting the muscular attachment in the occipital bone. Then, a 27-gauge needle was carefully inserted into the cisterna magna. After withdrawal of 0.2±0.3 ml CSF, an equal amount of fresh autologous blood, obtained from a femoral artery was, injected into the cisterna magna over a 3-min period. The needle was then withdrawn and the skin incision was stitched together. In order to allow an optimal blood distribution around basal intracranial arteries, the rat was kept in a 20 head down position for 30 min after the injection. Sham-operated animals (n 3) used for normal caliber of rat basilar arteries, underwent the atlanto-occipital membrane exposure only. The animals were allowed to recover from anesthesia and given free access to food and water. None of the rats showed signs of distress or pain during the post-operative period. The rats were reanesthetised at 48 h after the initial injection of autologous blood and then given a second injection of blood as mentioned above. Twenty rats were randomly assigned to either a control group or a treated group. Then, either 0.417 mg of classical liposome-entrapped Fasudil (treated group, n 10) or drug-free liposomes (control group, n 10) was applied to cisterna magna with a 27 gauge needle 1 h after a second blood injection. The drug-free liposomes contained the same amount of PL as the liposome-entrapped Fasudil. After the procedures, the wound was stitched together and the animals were treated as mentioned above.
buffered saline, perfusion fixation was performed with 2% glutaraldehyde at a perfusion pressure of 100 cm H2O. The brain with the basilar artery and arteries of circle of Willis was removed immediately and was fixed in 2% glutaraldehyde over 1 week. After appropriate fixation and paraffin embedding, rat basilar artery was continuously sliced and stained with hematoxylin eosin. For light microscopic evaluation, a basilar artery was divided into three portions (upper, middle, and lower part of the basilar artery). The caliber of each sample taken from each portion of basilar artery was measured and the value was expressed as the average of three portions. We measured the most spastic portion of each artery at 7 days and the measurement was performed three times in a double-blind fashion. Finally, the caliber of each sample was expressed as a percentage of the caliber of the normal basilar artery. Measurement of Fasudil concentration in CSF and blood At the time of sacrifice, 0.2 ± 0.4 ml of CSF was obtained from the cisterna magna of the animals and 5 ml of blood was taken for the measurement of Fasudil concentrations 7 days after SAH. Fasudil contained in the CSF and blood samples was quantified by high performance liquid chromatography (HPLC) (Beckman System Gold1, Beckman Instruments Inc., Fullerton, CA, USA). Each sample (50 ml) was injected onto a Alltech Spherisorb ODS±25 micron column (25 cm 4.6 mm) (Alltech Associates, Inc., Deerfield, IL, USA). The column was run using isocratic eluent conditions (30% acetonitrile in H2O, 0.05% TCA) and a flow rate of 1 ml/min. Fasudil eluted at 5.2 min as detected by its ultraviolet absorbance at 320 nm using a Beckman 166 UV detector. Fasudil was quantified by comparing the peak area of Fasudil samples to a standard Fasudil curve. STATISTICS All data in this study were expressed as mean standard deviation (SD). A two-tailed, paired Student's t-test was used to compare the difference between the values obtained before and after treatment. A probability value of <0.05 was considered to indicate a significant difference. RESULTS None of the rats showed signs of distress or pain during the post-operative period and all animals in this series survived the two blood injections into the cisterna magna. No rats were observed to develop neurologic deficits. Releasing property of liposome-entrapped Fasudil in vitro The loading efficiency of Fasudil was greater than 95% at a PL : Fasudil ratio of 1 : 0.4 (w/w), and liposomes routinely contained Fasudil at a concentration of 140±160 mg Fasudil/ mmol PL (0.43±0.49 mmol Fasudil/mmol PL). The release of Fasudil from classical liposomes in CSF was biphasic, with a T1/2a of 2.5 hours and a T1/2b of 355 hours (n=3). By contrast, Stealth liposomes were extremely stable in control CSF (n=3). The release of Fasudil from Stealth liposomes was primarily monophasic with a T1/2 of 16 weeks (Fig. 1).
Evaluation of vasoconstriction on basilar artery
Evaluation of vasoconstriction on basilar artery
On day 7 following the initial blood injection, the rats were deeply anesthetised with overdoses of ketamine and xylazine. After perfusion via a cardiac catheter with 300 ml of phosphate
The rats were given two injections, 48 h apart, of autologous arterial blood into the cisterna magna and this successfully induced delayed vasoconstriction of rat basilar artery (Fig. 2).
Journal of Clinical Neuroscience (2001) 8(6), 557±563
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Intrathecal application with liposome-entrapped Fasudil 559
% of Fasudil in liposomes
120 100 80 60 40 20 0 0
50
100 Time in h
150
Fig. 1 Graph showing the cumulative percentage of Fasudil released from classical liposomes (&) and Stealth liposomes () into control CSF in vitro at pH 7.4 and 37 C as a function of time (n 3). Over 7 days, 60% of the contents are released in classical liposomes, but by contrast, 95% of Fasudil still remains in Stealth liposomes. Vertical bars represent SD. In terms of classical liposomes, T1/2a of 2.5 h and T1/2b of 355 h represent the half life of Fasudil release in phase and the half-life of Fasudil release in b phase, respectively.
In the current study, liposomal Fasudil, at the non-toxic dose of 0.417 mg/rat, significantly prevented vasoconstriction in the rat basilar artery when compared to that of the control group (87.7 6.1% vs 66.3 9.8%, respectively, ***P < 0.001, Fig. 3). As Stealth liposomes only released 5% of Fasudil over 7 days of an in vitro leakage study, an animal study was not carried out on Stealth liposomes. Concentration of liposome-entrapped Fasudil in CSF and blood Just before sacrifice, CSF and blood were collected from the animals for measurement of Fasudil concentrations by HPLC. We were unable to collect CSF from three rats in the treated group, due to technical difficulties. Based on the administered dose of Fasudil of 4.17 mg/rat, and an average of 1.0 ml of CSF per 300 to 400 g rat, the initial concentration of liposomal Fasudil would be 417 mg/ml. The concentration of liposome-entrapped Fasudil in CSF 7 days after SAH was 0.98 0.69 mg/ml (n 7). As expected, no Fasudil was detectable in blood samples. DISCUSSION The main observation of the current study is that intrathecal administration of liposomal Fasudil significantly reduced the severity of narrowing in basilar artery and produced no obvious adverse effect in the rat SAH model. The advantage of using liposomes as drug carriers in the central nervous system comes from the ability to achieve a sustained release of therapeutically relevant concentrations of therapeutic drugs directly into the CSF over a period of days after a single intrathecal administration of the liposomal drug. The relatively slow release of drug from liposomes mimicks some aspects of drug infusion, including a significant decrease in the toxicity of the free drug. Thus, the therapeutic index of Fasudil was increased by virture of both an increase efficacy and a decrease in systemic toxicity. Intrathecal vasodilatory therapy for the treatment of cerebral vasospasm could minimise the risk of systemic adverse effects such as hypotension. Therefore, intrathecal administration of calciumantagonistshasbeenchosenandevaluatedforprevention of cerebral vasospasm in dogs.5,6 However, externalised ventricular catheter or repeated puncture must be needed to maintain a therapeutic drug concentration in the CSF. Frequent or continuous intrathecal administration is impractical, especially in a & 2001 Harcourt Publishers Ltd
Fig. 2 Light microscopic evaluation of rat basilar artery on 7 days after SAH. When compared to normal caliber obtained from sham-operated group (upper), basilar artery in control group reveals not only marked vasoconstriction but also endothelial corrugation (lower). On the contrary, treatment with liposomal Fasudil substantially prevents vasoconstriction on rat basilar artery (middle). Scale bar indicates 100 mm. (Hematoxylin eosin, magnification 200).
clinical setting. Therefore, a single injection of liposomal Fasudil whichcanbesafelydeliveredintheCSFmightbeanovelapproach for the treatment of cerebral vasospasm following SAH. The drug release rate from the liposomes is dependent on various conditions, including the liposome composition and size, the physicochemical properties of the drug, and the method of drug loading into liposomes.11 As expected, Stealth liposome resulted in only 5 % of the drug release over 7 days. Therefore, classical liposomes were selected, but Stealth liposomes were not verified for the animal study. It has been clinically reported that Fasudil hydrochloride shows a Journal of Clinical Neuroscience (2001) 8(6), 557±563
560 Takanashi et al.
Sham-operated (n =3) †
Treated (n =10) *** †
Control (n =10) 0
25
50
75
100
(%) Fig. 3 Bar graph showing the average percentage of caliber on rat basilar artery. When normal caliber on basilar artery obtained from sham-operated group is expressed as 100%, caliber on basilar artery of the treatment group is 87.7 6.1% and that of the control group is 66.3 9.8%. Number in parenthesis indicates number of animals measured in each group. (Mean SD, ***P < 0.001 vs treated group, yP < 0.001 vs Sham-operated group).
significant reduction in angiographically revealed vasospasm in patients with SAH at the dose of 1.0±1.5 mg/kg/day.4 According to clinical data as well as the leakage figure (Fig. 1), we chose a dose of 0.417 mg of Fasudil in classical liposomes. In the current study, injection of liposomal Fasudil resulted in a CSF concentration of 0.98 0.69 mg/ml at 7 days after SAH in rats. This result suggested that most of the Fasudil was released from classical liposomes in vivo in rat CSF in 7 days, while approximately 60% was released in vitro in control CSF. Therefore, between 0.20 and 0.23 mg/kg/day of released Fasudil can significantly prevent vasoconstriction in a rat SAH model. This dose is only 13% to 20% of the corresponding intravenous dosage range in humans of 1.0±1.5 mg/kg/day. It has been widely accepted that interaction with blood components and the mononuclear phagocyte system are responsible for the clearance and elimination of liposomes following intravenous application.12±14 In the case of SAH, CSF contains a small amount of blood compared to blood stream. Therefore, it is conceivable that blood components in the CSF might be accountable for accelerating the drug release from liposomes in vivo. However, as the clearance and elimination of liposomes in the CSF are little understood, further experiments will be required to clarify the optimal drug release rate as well as circulation of liposomes in the CSF. In the current study, encapsulation of Fasudil in classical liposomes changes the pharmacokinetics of the free drug and provides sustained-release of the drug with a T1/2a of 2.5 hours and a terminal half-life of 355 hours. By comparison, free Fasudil is cleared quickly from the blood with a half-life of less than 15 min.4 Thus, even if the free drug were administered by the intrathecal route, redistribution of the drug would quickly reduce its concentration below the therapeutic range. The release rate for the liposomal drug appears to be appropriate for the danger period for cerebral vasospasm. Fasudil is considered to be an intracellular calcium antagonist, by inhibiting cyclic nucleotide-dependent protein kinases and the calcium/calmodulin-dependent MLCK.15±18 It has also been reported that Fasudil by intravenous administration in a canine SAH model increases the blood flow to the cerebral cortex by a greater vasodilatory effect on parenchymal arterioles.19,20 In the current study, intrathecal application with sustained-drug release in the CSF may enhance the vasodilating effect of Fasudil because direct application into the CSF allows diffuse distribution of the drug through the entire neuraxis. Although Fasudil reduced angiographically verified cerebral vasospasm and produced a modest increase in cerebral blood flow in a dose-dependent manner, higher doses of Fasudil were Journal of Clinical Neuroscience (2001) 8(6), 557±563
inclined to cause systemic adverse effects such as hypotension.21,22 However, as liposomal Fasudil small enough to be applied in the CSF could continuously deliver drug into the brain parenchyma, the use of liposomal Fasudil could be advantageous in comparison with peripheral administration. In conclusion, treatment of a rat SAH model with liposomal Fasudil by intrathecal injection has resulted in preventing vasoconstriction of basilar artery on 7 days without any adverse effects. Decreased doses of liposomal drug were required, as compared with peripherally administered drug, resulting in lower toxicities. The liposomal drug functioned as a sustained drug release system resulting in free drug concentration within the therapeutic range. The promising results obtained from the current animal study may have the potential to lead to a new approach in the treatment of cerebral vasospasm following SAH. ACKNOWLEDGEMENTS This work is supported in part by a grant from Yokohama Foundation for Advancement of Medical Science in Japan to Dr Y. Takanashi, MRC grant MT-9127 to Dr T. Allen, and the American Heart Association Bugher Foundation for Stroke Award to Dr J. Zhang. REFERENCES 1.
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Adams H, Kassel N, Torner J, Haley E. Predicting outcome ischemia after aneurysmal subarachnoid hemorrhage: influences of clinical condition, CT results and antifiblinolytic therapy: a report of the Cooperative Aneurysm Study. Neurology 1987; 37: 1586±1591. Solenski N, Haley E, Kassel N, et al. Medical complications of aneurysmal subarachnoid hemorrhage: a report of multicenter, cooperative aneurysm study. Crit Care Med 1995; 23: 1007±1017. Nakashima S, Tabuchi K, Shimokawa S, Fukuyama K, Mineta T, Abe M. Combination therapy of Fasudil hydrochloride and ozagrel sodium for cerebral vasospasm following aneurysmal subarachnoid hemorrhage. Neurol Med Chir (Tokyo) 1998; 38: 805±811. Shibuya M, Suzuki Y, Sugita K, et al. Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage. J Neurosurg 1992; 76: 571±577. Gioia A, White R, Bakhtian B, Robertson J. Evaluation of the efficacy of intrathecal nimodipine in canine models of chronic cerebral vasospasm. J Neurosurg 1985; 62: 721±728. Voldby B, Petersen O, Buhl M, Jakobsen P, éstergaard R. Reversal of cerebral arterial spasm by intrathecal administration of a calcium antagonist (nimodipine). An experimental study. Acta Neurochir (Wien) 1984; 70: 243±254. Bolotin E, Cohen R, Bar L, Emanuel S, Lasic D, Barenholz Y. Ammonium sulphate gradients for efficient and stable remote loading of amphipathic weak bases into liposomes and ligandosomes. J Liposome Res 1994; 4: 455±479. Bartlett G. Phosphorus assay in column chromatography. J Biol Chem 1959; 234: 466±468. Delgado T, Brismar J, Svendgaard N. Subarachnoid haemorrhage in the rat: angiography and fluorescence microscopy of the major cerebral arteries. Stroke 1985; 16: 595±602. Suzuki H, Kanamaru K, Tsunoda H, et al. Heme oxygenase-1 induction as an intrinsic regulation against delayed cerebral vasospasm in rats. J Clin Invest 1999; 104: 59±66. Allen T, Hansen C, Lopes de Menezes D. Pharmacokinetics of long circulating liposomes. Adv Drug Del Rev 1995; 16: 267±284. Bonte F, Juliano R. Interactions of liposomes with serum proteins. Chem Phys Lipids 1986; 40: 359±372. Devine D, Marjan J. The role of immunoproteins in the survival of liposomes in the circulation. Crit Rev Ther Drug Carrier Syst 1997; 14: 105±131. Jones M, Nicholas A. The effect of blood serum on the size and stability of phospholipid liposomes. Biochim Biophys Acta 1991; 1065: 145±152. Asano T, Ikegaki I, Satoh S, et al. Mechanism of action of a novel antivasospasm drug, HA1077. J Pharmacol Exp Ther 1987; 241: 1033±1040. Asano T, Suzuki T, Tsuchiya M, et al. Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase. Br J Pharmacol 1989; 98: 1091±1100.
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Neuronal free Ca2+ and BBB permeability and ultrastructure in head injury with secondary insult 17.
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Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 1984; 23: 5036±5041. Seto M, Sasaki Y, Sasaki Y, Hidaka H. Effects of HA 1077, a protein kinase inhibitor, on myosin phosphorylation and tension in smooth muscle. Eur J Pharmacol 1991; 195: 267±272. Satoh S, Suzuki Y, Harada T, et al. Possible prophylactic potential of HA 1077, a Ca2+ antagonist and vasodilator, on chronic cerebral vasospasm. Eur J Pharmacol 1992; 220: 243±248.
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Takayasu M, Dacey R. Effect of HA 1077, a novel calcium antagonistic spasmolytic agent on intracerebral arterioles of rats. Acta Neurochir (Wien) 1990; 103: 67±70. Sako K, Tsuchiya M, Yonemasu M, Asano T. HA 1077 a novel calcium antagonistic antivasospasm drug, increases both cerebral blood flow and glucose metabolism in conscious rats. Eur J Pharmacol 1991; 209: 39±43. Shibuya M, Suzuki Y, Takayasu M, et al. The effect of intracellular calcium antagonist HA 1077 on delayed cerebral vasospasm in dogs. Acta Neurochir (Wien) 1988; 90: 53±59.
Journal of Clinical Neuroscience (2001) 8(6), 561±563 & 2001 Harcourt Publishers Ltd DOI: 10.1054/jocn.2001.0980, available online at http://www.idealibrary.com on
Neuronal free Ca2+ and BBB permeability and ultrastructure in head injury with secondary insult Fei Zhou PHD, Zhang Xiang PHD, Wang Xiao Feng, Liu Xian Zhen Department of Neurosurgery, Xijing Hospital, Xian, P.R. China
Summary Objective: To study changes in free calcium (Ca2), neuronal and blood±brain barrier (BBB) permeability and ultrastructure in brain after diffuse axonal injury (DAI) with secondary brain insults (SBIs). Method: One hundred and twenty Sprague-Dawley (SD) rats were randomised into control, DAI alone and DAI with SBI groups which were sub-divided into 5 groups that were 0.5 h, 2 h, 12 h, 24 h, 48 h post trauma. The animal models of DAI and DAI with SBI have been described before (2). Fluorescence probe Fluo-3/Am was used to measure free Ca2 in neurons. Laser scan microscopy was used to detect fluorescence intensity. After the animals were anesthetized, Lanthanum nitrate liquid was used for intracardiac perfusion to assess BBB permeability. Under the transmission electron microscope, changes in cerebral ultrastructure and BBB permeability were observed. Results: The fluorescence intensity was weak in the control. The concentration of free Ca2 in neurons was obviously increased at 30 min after brain injury, reached a peak at 12 h±24 h (P < 0.01), and appeared to decrease at 48 h after injury. In the DAI alone group, BBB tight junction opening with particles of Lanthanum nitrate outside the vessels was found at 30min after injury, and peaked at 24 h. In DAI with SBI, the changes in ultrastructure and BBB permeability were more severe than that in the DAI alone group at the same time interval. The shape of the fluorescence concentration curve was basically the same for both kinds of brain injury. The intensity of fluorescence in DAI with SBI was higher than that in the DAI alone group at the same time interval (P < 0.05). Conclusion: In DAI alone and DAI with SBI, Ca2 overload and BBB permeability changes interact and both play important roles in the aggravation of brain damage. & 2001 Harcourt Publishers Ltd Keywords: brain, diffuse axonal injury (DAI), secondary brain insult (SBI), calcium, blood±brain barrier (BBB)
INTRODUCTION Brain diffuse axonal injury (DAI) is associated with high morbidity and mortality.1 In a previous study, we successfully established a rat DAI model by lateral rotation of the head, observing the pathological changes of macroscopic petechiae. Additionally at the frontal and temporal bases, axonal retraction balls (ARBs) were observed microscopically, scattered in the brain and brainstem.2 Our previous study on brain thromboxane A2 (TXA2) and prostoglandin I2 (PGI2) levels in diffuse brain injury alone and with hypotension indicated that PGI2 may act as a repair mechanism by providing energy and precursors to the injured tissue and the production of vasoactive arachidonic acid products, especially TXA2, is closely connected to the severity of brain damage.3 Pyrexia, another common symptom seen in multiple-trauma patients with head injury may also worsen the outcome.4 The present study was designed to investigate the changes of neuronal free Ca2 and Received 14 April 2000 Accepted 19 July 2000 Correspondence to: Fei Zhou, Department of Neurosurgery, Xijing Hospital, Xi'an 710032, P. R. China. Tel.: 86 29 2534570; Fax: 86 29 3249273; E-mail: [email protected]
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BBB permeability and ultrastructure in experimental head injury with a pyrexial secondary brain insult (SBI). MATERIAL AND METHODS 1. Animal grouping and model: One hundred and twenty healthy male SD rats, weighing 25020 g, were randomised into control, DAI alone and DAI with SBI groups which were sub-divided into five groups that were 0.5 h, 2 h, 12 h, 24 h, 48 h post trauma. In a previous study, we had successfully produced diffuse axonal injury in rats.2 The same model was used in the present study. All rats were anesthetized with peritoneal injection of 1% sodium pentobarbital (30 mg/kg).The rat's head was horizontally secured to the injury device by two lateral ear bars, a head clip and an anterior teeth hole, with its body 20 oblique to the top of laboratory table. In the injury group, a trigger was pressed and the device instantly rotated the head through a 90 angle in the lateral plane. It had been identified previously that the head rotation was finished in less than 30 milliseconds with an angular velocity above 761 rad/s and an angular acceleration above 1.87 105 rad/s2. In the control group each rat was released from the device once secured. Acute DAI with pyrexia was achieved as Journal of Clinical Neuroscience (2001) 8(6), 557±563
Laboratory studies
Intrathecal application with liposome-entrapped Fasudil for cerebral vasospasm following subarachnoid hemorrhage in rats Yoshihiro Takanashi1 MD PHD, Tatsuhiro Ishida2 PHD, Toshinari Meguro3 MD, Marc J. Kirchmeier2 PHD, Theresa M. Allen2 PHD, John H. Zhang3 MD PHD 1
Department of Neurosurgery, Yokohama City University, Yokohama, Japan, 2Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, 3Department of Neurosurgery, University of Mississippi Medical Center, Jackson, Mississippi, USA
Summary To date, the pharmacological approach to cerebral vasospasm following subarachnoid hemorrhage has been hampered in part by an inability to attain sufficiently high concentrations of vasodilator drugs in the cerebrospinal fluid (CSF). To overcome this limitation of current drug therapy, we have developed a sustained-release preparation of protein kinase inhibitor Fasudil. Cerebral vasospasm in rats was induced by double-injection method. Treated rats received 0.417 mg liposome-entrapped Fasudil via the cisterna magna and control rats received drug-free liposomes in the same manner. The diameter of the basilar artery was assessed at 7 days after the initial blood injection. Vasoconstriction of the rat basilar artery was significantly reduced in group treated with liposomal Fasudil compared to the control group (treated group: 87.7 6.18%, n 10; control group: 66.3 9.82%, n 10; ***P < 0.001). This new approach for cerebral vasospasm may have significant potential for use in the clinical setting. & 2001 Harcourt Publishers Ltd Keywords: cerebral vasospasm, drug delivery systems, Fasudil, liposomes, rat, subarachnoid hemorrhage
INTRODUCTION Cerebral arterial vasospasm with delayed ischemic neurological deficit occurs in one-third of patients with aneurysmal subarachnoid hemorrhage (SAH).1 Despite considerable advances in perioperative management of these patients, including the use of calcium antagonists, cerebral vasospasm, which is the delayed narrowing of major arteries at the base of the brain, still remains a major cause of morbidity and mortality after SAH.2 Recently, Fasudil hydrochloride (1-5-isoquinolinesulphonylhomopiperazine), which has a vasodilating effect, has been used in patients with SAH and encouraging data have been reported for prevention of cerebral vasospasm.3,4 Experimental work has repeatedly shown that vasodilator drugs can reverse established angiographically identified vasospasm when administered by the intrathecal route, despite being ineffective when administered by the intravenous or intra-arterial route.5,6 Although intrathecal vasodilatory therapy for the treatment of cerebral vasospasm could minimise the risk of systemic adverse effects such as hypotension, it is difficult to continuously maintain a therapeutic drug concentration in the CSF. The limited application of intrathecal route, especially in clinical studies, is associated with the troublesome procedure and sideeffects such as meningitis as the result of long-term indwelling catheters. Moreover, the time during which drug concentrations remain in the therapeutic window may be transient even if bolus application by the intrathecal route was feasible. We have devised a novel method for the prevention of experimental vasospasm by which a sustained-release form of the drug can maintain a therapeutic concentration in the CSF within Received 28 August 2000 Accepted 31 October 2000 Correspondence to: Yoshihiro Takanashi MD, PhD, Department of Neurosurgery, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Japan, 236-0004. Tel.: 81 45 787-2663; Fax: 81 45 783 6121; E-mail: [email protected]
a therapeutic window. The aim of the present study is to evaluate whether intrathecal administration of the liposomal Fasudil can prevent cerebral vasospasm following SAH in a rat model. MATERIALS AND METHODS Preparation of liposomes Liposomes were made with two different lipid compositions. Classical liposomes were composed of hydrogenated soy phosphatidylcholine : cholesterol (HSPC : CHOL) at 2 : 1 molar ratio. Stealth liposomes were composed of HSPC : CHOL : methoxypolyethyleneglycol (Mr 2000) covalently linked to distearoylphosphatidylethanolamine (mPEG2000-DSPE) at 2 : 1 : 0.1 molar ratio. For Fasudil-loaded liposomes, we used remote loading with an ammonium sulfate gradient to encapsulate the drug.7 Dried lipid films were hydrated in 250 mM ammonium sulfate (pH 3.0). Then, extrusion (Lipex Biomembranes Extruder, Vancouver, British Columbia, Canada) was sequentially carried out to produce homogeneously sized liposome preparations, using a series of polycarbonate filters (Nuclepore Corp., Pleasanton, CA, USA) with pore sizes ranging from 0.4 to 0.08 mm. The mean diameter of liposomes which was determined by dynamic light scattering using a Brookhaven B190 submicron particle size analyzer (Brookhaven Instruments Corp., Holtsville, NY, USA) was in the range of 110 10 nm. The external buffer was then exchanged by eluting through a Sephadex G50 column equilibrated with 10% sucrose (pH 8.0). Fasudil (Sigma Chemical Co., St Louis, MO, USA) was loaded into the liposomes at a phospholipid (PL): Fasudil ratio of 1 : 0.4 (w/w) and incubated for 1 h at 65 C. Liposome-entrapped Fasudil was separated from free Fasudil using a Sephadex G50 column eluted with 25 mM N-2-hydroxyethyl piperazine-N 0 -2-ethansulfonic acid (HEPES) and 140 mM NaCl buffer (pH 7.4). Spectrophotometry ( 320 nm) finally determined the concentration of the liposome-entrapped Fasudil, and PL concentrations were determined by means of the Bartlett colorimetric assay.8 The loading efficiency of Fasudil into liposomes was also determined. 557
558 Takanashi et al.
Releasing property of liposome-entrapped Fasudil in vitro The in vitro drug-release kinetics of the liposome-entrapped Fasudil was measured in control CSF (Sigma Chemical Co, St Louis, MO, USA). Either classical liposomes or Stealth liposomes (0.8 ml) containing 0.417 mg of Fasudil and 1.303 mmol PL were incubated in 4.2 ml of CSF at 37 C for 7 days. Periodically, 0.5 ml of the medium was withdrawn and replaced with an equivalent volume of fresh CSF to mimic CSF turnover in vivo. Free Fasudil was separated from liposome-entrapped Fasudil using a Sephadex CL-4B column equilbrated with HEPES buffer (pH 7.4), as above. Following dissolution in 100% methanol, the concentration of the Fasudil in liposomes was then determined by spectrophotometry (=320 nm), and PL concentrations were determined using the Bartlett colorimetric assay.8 Experimental model of SAH and study design Male Sprague±Dawley rats weighing 350±400 g were used for the experiments. Animals were housed in the vivarium with free access to water and food in a 12-h, day±night cycle. Approval from the institutional ethics committee (University of Mississippi Medical Center) was obtained for all procedures. SAH in a rat was induced by injection of autologous arterial blood into the cisterna magna twice.9,10 Rats were anethsthetised by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and allowed to breathe spontaneously. Anesthesia was maintained by additional injections of ketamine and xylazine as needed. Rectal temperature was maintained at approximately 37 0.5 C throughout the surgical procedure using a heating pad and overhead lamp. Under sterile condition, rats were placed in a head flexion angle of 30 with a rolled-up gauze under the neck. The atlanto-occipital membrane was fully exposed through a midline skin incision while dissecting the muscular attachment in the occipital bone. Then, a 27-gauge needle was carefully inserted into the cisterna magna. After withdrawal of 0.2±0.3 ml CSF, an equal amount of fresh autologous blood, obtained from a femoral artery was, injected into the cisterna magna over a 3-min period. The needle was then withdrawn and the skin incision was stitched together. In order to allow an optimal blood distribution around basal intracranial arteries, the rat was kept in a 20 head down position for 30 min after the injection. Sham-operated animals (n 3) used for normal caliber of rat basilar arteries, underwent the atlanto-occipital membrane exposure only. The animals were allowed to recover from anesthesia and given free access to food and water. None of the rats showed signs of distress or pain during the post-operative period. The rats were reanesthetised at 48 h after the initial injection of autologous blood and then given a second injection of blood as mentioned above. Twenty rats were randomly assigned to either a control group or a treated group. Then, either 0.417 mg of classical liposome-entrapped Fasudil (treated group, n 10) or drug-free liposomes (control group, n 10) was applied to cisterna magna with a 27 gauge needle 1 h after a second blood injection. The drug-free liposomes contained the same amount of PL as the liposome-entrapped Fasudil. After the procedures, the wound was stitched together and the animals were treated as mentioned above.
buffered saline, perfusion fixation was performed with 2% glutaraldehyde at a perfusion pressure of 100 cm H2O. The brain with the basilar artery and arteries of circle of Willis was removed immediately and was fixed in 2% glutaraldehyde over 1 week. After appropriate fixation and paraffin embedding, rat basilar artery was continuously sliced and stained with hematoxylin eosin. For light microscopic evaluation, a basilar artery was divided into three portions (upper, middle, and lower part of the basilar artery). The caliber of each sample taken from each portion of basilar artery was measured and the value was expressed as the average of three portions. We measured the most spastic portion of each artery at 7 days and the measurement was performed three times in a double-blind fashion. Finally, the caliber of each sample was expressed as a percentage of the caliber of the normal basilar artery. Measurement of Fasudil concentration in CSF and blood At the time of sacrifice, 0.2 ± 0.4 ml of CSF was obtained from the cisterna magna of the animals and 5 ml of blood was taken for the measurement of Fasudil concentrations 7 days after SAH. Fasudil contained in the CSF and blood samples was quantified by high performance liquid chromatography (HPLC) (Beckman System Gold1, Beckman Instruments Inc., Fullerton, CA, USA). Each sample (50 ml) was injected onto a Alltech Spherisorb ODS±25 micron column (25 cm 4.6 mm) (Alltech Associates, Inc., Deerfield, IL, USA). The column was run using isocratic eluent conditions (30% acetonitrile in H2O, 0.05% TCA) and a flow rate of 1 ml/min. Fasudil eluted at 5.2 min as detected by its ultraviolet absorbance at 320 nm using a Beckman 166 UV detector. Fasudil was quantified by comparing the peak area of Fasudil samples to a standard Fasudil curve. STATISTICS All data in this study were expressed as mean standard deviation (SD). A two-tailed, paired Student's t-test was used to compare the difference between the values obtained before and after treatment. A probability value of <0.05 was considered to indicate a significant difference. RESULTS None of the rats showed signs of distress or pain during the post-operative period and all animals in this series survived the two blood injections into the cisterna magna. No rats were observed to develop neurologic deficits. Releasing property of liposome-entrapped Fasudil in vitro The loading efficiency of Fasudil was greater than 95% at a PL : Fasudil ratio of 1 : 0.4 (w/w), and liposomes routinely contained Fasudil at a concentration of 140±160 mg Fasudil/ mmol PL (0.43±0.49 mmol Fasudil/mmol PL). The release of Fasudil from classical liposomes in CSF was biphasic, with a T1/2a of 2.5 hours and a T1/2b of 355 hours (n=3). By contrast, Stealth liposomes were extremely stable in control CSF (n=3). The release of Fasudil from Stealth liposomes was primarily monophasic with a T1/2 of 16 weeks (Fig. 1).
Evaluation of vasoconstriction on basilar artery
Evaluation of vasoconstriction on basilar artery
On day 7 following the initial blood injection, the rats were deeply anesthetised with overdoses of ketamine and xylazine. After perfusion via a cardiac catheter with 300 ml of phosphate
The rats were given two injections, 48 h apart, of autologous arterial blood into the cisterna magna and this successfully induced delayed vasoconstriction of rat basilar artery (Fig. 2).
Journal of Clinical Neuroscience (2001) 8(6), 557±563
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Intrathecal application with liposome-entrapped Fasudil 559
% of Fasudil in liposomes
120 100 80 60 40 20 0 0
50
100 Time in h
150
Fig. 1 Graph showing the cumulative percentage of Fasudil released from classical liposomes (&) and Stealth liposomes () into control CSF in vitro at pH 7.4 and 37 C as a function of time (n 3). Over 7 days, 60% of the contents are released in classical liposomes, but by contrast, 95% of Fasudil still remains in Stealth liposomes. Vertical bars represent SD. In terms of classical liposomes, T1/2a of 2.5 h and T1/2b of 355 h represent the half life of Fasudil release in phase and the half-life of Fasudil release in b phase, respectively.
In the current study, liposomal Fasudil, at the non-toxic dose of 0.417 mg/rat, significantly prevented vasoconstriction in the rat basilar artery when compared to that of the control group (87.7 6.1% vs 66.3 9.8%, respectively, ***P < 0.001, Fig. 3). As Stealth liposomes only released 5% of Fasudil over 7 days of an in vitro leakage study, an animal study was not carried out on Stealth liposomes. Concentration of liposome-entrapped Fasudil in CSF and blood Just before sacrifice, CSF and blood were collected from the animals for measurement of Fasudil concentrations by HPLC. We were unable to collect CSF from three rats in the treated group, due to technical difficulties. Based on the administered dose of Fasudil of 4.17 mg/rat, and an average of 1.0 ml of CSF per 300 to 400 g rat, the initial concentration of liposomal Fasudil would be 417 mg/ml. The concentration of liposome-entrapped Fasudil in CSF 7 days after SAH was 0.98 0.69 mg/ml (n 7). As expected, no Fasudil was detectable in blood samples. DISCUSSION The main observation of the current study is that intrathecal administration of liposomal Fasudil significantly reduced the severity of narrowing in basilar artery and produced no obvious adverse effect in the rat SAH model. The advantage of using liposomes as drug carriers in the central nervous system comes from the ability to achieve a sustained release of therapeutically relevant concentrations of therapeutic drugs directly into the CSF over a period of days after a single intrathecal administration of the liposomal drug. The relatively slow release of drug from liposomes mimicks some aspects of drug infusion, including a significant decrease in the toxicity of the free drug. Thus, the therapeutic index of Fasudil was increased by virture of both an increase efficacy and a decrease in systemic toxicity. Intrathecal vasodilatory therapy for the treatment of cerebral vasospasm could minimise the risk of systemic adverse effects such as hypotension. Therefore, intrathecal administration of calciumantagonistshasbeenchosenandevaluatedforprevention of cerebral vasospasm in dogs.5,6 However, externalised ventricular catheter or repeated puncture must be needed to maintain a therapeutic drug concentration in the CSF. Frequent or continuous intrathecal administration is impractical, especially in a & 2001 Harcourt Publishers Ltd
Fig. 2 Light microscopic evaluation of rat basilar artery on 7 days after SAH. When compared to normal caliber obtained from sham-operated group (upper), basilar artery in control group reveals not only marked vasoconstriction but also endothelial corrugation (lower). On the contrary, treatment with liposomal Fasudil substantially prevents vasoconstriction on rat basilar artery (middle). Scale bar indicates 100 mm. (Hematoxylin eosin, magnification 200).
clinical setting. Therefore, a single injection of liposomal Fasudil whichcanbesafelydeliveredintheCSFmightbeanovelapproach for the treatment of cerebral vasospasm following SAH. The drug release rate from the liposomes is dependent on various conditions, including the liposome composition and size, the physicochemical properties of the drug, and the method of drug loading into liposomes.11 As expected, Stealth liposome resulted in only 5 % of the drug release over 7 days. Therefore, classical liposomes were selected, but Stealth liposomes were not verified for the animal study. It has been clinically reported that Fasudil hydrochloride shows a Journal of Clinical Neuroscience (2001) 8(6), 557±563
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Sham-operated (n =3) †
Treated (n =10) *** †
Control (n =10) 0
25
50
75
100
(%) Fig. 3 Bar graph showing the average percentage of caliber on rat basilar artery. When normal caliber on basilar artery obtained from sham-operated group is expressed as 100%, caliber on basilar artery of the treatment group is 87.7 6.1% and that of the control group is 66.3 9.8%. Number in parenthesis indicates number of animals measured in each group. (Mean SD, ***P < 0.001 vs treated group, yP < 0.001 vs Sham-operated group).
significant reduction in angiographically revealed vasospasm in patients with SAH at the dose of 1.0±1.5 mg/kg/day.4 According to clinical data as well as the leakage figure (Fig. 1), we chose a dose of 0.417 mg of Fasudil in classical liposomes. In the current study, injection of liposomal Fasudil resulted in a CSF concentration of 0.98 0.69 mg/ml at 7 days after SAH in rats. This result suggested that most of the Fasudil was released from classical liposomes in vivo in rat CSF in 7 days, while approximately 60% was released in vitro in control CSF. Therefore, between 0.20 and 0.23 mg/kg/day of released Fasudil can significantly prevent vasoconstriction in a rat SAH model. This dose is only 13% to 20% of the corresponding intravenous dosage range in humans of 1.0±1.5 mg/kg/day. It has been widely accepted that interaction with blood components and the mononuclear phagocyte system are responsible for the clearance and elimination of liposomes following intravenous application.12±14 In the case of SAH, CSF contains a small amount of blood compared to blood stream. Therefore, it is conceivable that blood components in the CSF might be accountable for accelerating the drug release from liposomes in vivo. However, as the clearance and elimination of liposomes in the CSF are little understood, further experiments will be required to clarify the optimal drug release rate as well as circulation of liposomes in the CSF. In the current study, encapsulation of Fasudil in classical liposomes changes the pharmacokinetics of the free drug and provides sustained-release of the drug with a T1/2a of 2.5 hours and a terminal half-life of 355 hours. By comparison, free Fasudil is cleared quickly from the blood with a half-life of less than 15 min.4 Thus, even if the free drug were administered by the intrathecal route, redistribution of the drug would quickly reduce its concentration below the therapeutic range. The release rate for the liposomal drug appears to be appropriate for the danger period for cerebral vasospasm. Fasudil is considered to be an intracellular calcium antagonist, by inhibiting cyclic nucleotide-dependent protein kinases and the calcium/calmodulin-dependent MLCK.15±18 It has also been reported that Fasudil by intravenous administration in a canine SAH model increases the blood flow to the cerebral cortex by a greater vasodilatory effect on parenchymal arterioles.19,20 In the current study, intrathecal application with sustained-drug release in the CSF may enhance the vasodilating effect of Fasudil because direct application into the CSF allows diffuse distribution of the drug through the entire neuraxis. Although Fasudil reduced angiographically verified cerebral vasospasm and produced a modest increase in cerebral blood flow in a dose-dependent manner, higher doses of Fasudil were Journal of Clinical Neuroscience (2001) 8(6), 557±563
inclined to cause systemic adverse effects such as hypotension.21,22 However, as liposomal Fasudil small enough to be applied in the CSF could continuously deliver drug into the brain parenchyma, the use of liposomal Fasudil could be advantageous in comparison with peripheral administration. In conclusion, treatment of a rat SAH model with liposomal Fasudil by intrathecal injection has resulted in preventing vasoconstriction of basilar artery on 7 days without any adverse effects. Decreased doses of liposomal drug were required, as compared with peripherally administered drug, resulting in lower toxicities. The liposomal drug functioned as a sustained drug release system resulting in free drug concentration within the therapeutic range. The promising results obtained from the current animal study may have the potential to lead to a new approach in the treatment of cerebral vasospasm following SAH. ACKNOWLEDGEMENTS This work is supported in part by a grant from Yokohama Foundation for Advancement of Medical Science in Japan to Dr Y. Takanashi, MRC grant MT-9127 to Dr T. Allen, and the American Heart Association Bugher Foundation for Stroke Award to Dr J. Zhang. REFERENCES 1.
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Adams H, Kassel N, Torner J, Haley E. Predicting outcome ischemia after aneurysmal subarachnoid hemorrhage: influences of clinical condition, CT results and antifiblinolytic therapy: a report of the Cooperative Aneurysm Study. Neurology 1987; 37: 1586±1591. Solenski N, Haley E, Kassel N, et al. Medical complications of aneurysmal subarachnoid hemorrhage: a report of multicenter, cooperative aneurysm study. Crit Care Med 1995; 23: 1007±1017. Nakashima S, Tabuchi K, Shimokawa S, Fukuyama K, Mineta T, Abe M. Combination therapy of Fasudil hydrochloride and ozagrel sodium for cerebral vasospasm following aneurysmal subarachnoid hemorrhage. Neurol Med Chir (Tokyo) 1998; 38: 805±811. Shibuya M, Suzuki Y, Sugita K, et al. Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage. J Neurosurg 1992; 76: 571±577. Gioia A, White R, Bakhtian B, Robertson J. Evaluation of the efficacy of intrathecal nimodipine in canine models of chronic cerebral vasospasm. J Neurosurg 1985; 62: 721±728. Voldby B, Petersen O, Buhl M, Jakobsen P, éstergaard R. Reversal of cerebral arterial spasm by intrathecal administration of a calcium antagonist (nimodipine). An experimental study. Acta Neurochir (Wien) 1984; 70: 243±254. Bolotin E, Cohen R, Bar L, Emanuel S, Lasic D, Barenholz Y. Ammonium sulphate gradients for efficient and stable remote loading of amphipathic weak bases into liposomes and ligandosomes. J Liposome Res 1994; 4: 455±479. Bartlett G. Phosphorus assay in column chromatography. J Biol Chem 1959; 234: 466±468. Delgado T, Brismar J, Svendgaard N. Subarachnoid haemorrhage in the rat: angiography and fluorescence microscopy of the major cerebral arteries. Stroke 1985; 16: 595±602. Suzuki H, Kanamaru K, Tsunoda H, et al. Heme oxygenase-1 induction as an intrinsic regulation against delayed cerebral vasospasm in rats. J Clin Invest 1999; 104: 59±66. Allen T, Hansen C, Lopes de Menezes D. Pharmacokinetics of long circulating liposomes. Adv Drug Del Rev 1995; 16: 267±284. Bonte F, Juliano R. Interactions of liposomes with serum proteins. Chem Phys Lipids 1986; 40: 359±372. Devine D, Marjan J. The role of immunoproteins in the survival of liposomes in the circulation. Crit Rev Ther Drug Carrier Syst 1997; 14: 105±131. Jones M, Nicholas A. The effect of blood serum on the size and stability of phospholipid liposomes. Biochim Biophys Acta 1991; 1065: 145±152. Asano T, Ikegaki I, Satoh S, et al. Mechanism of action of a novel antivasospasm drug, HA1077. J Pharmacol Exp Ther 1987; 241: 1033±1040. Asano T, Suzuki T, Tsuchiya M, et al. Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase. Br J Pharmacol 1989; 98: 1091±1100.
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Journal of Clinical Neuroscience (2001) 8(6), 561±563 & 2001 Harcourt Publishers Ltd DOI: 10.1054/jocn.2001.0980, available online at http://www.idealibrary.com on
Neuronal free Ca2+ and BBB permeability and ultrastructure in head injury with secondary insult Fei Zhou PHD, Zhang Xiang PHD, Wang Xiao Feng, Liu Xian Zhen Department of Neurosurgery, Xijing Hospital, Xian, P.R. China
Summary Objective: To study changes in free calcium (Ca2), neuronal and blood±brain barrier (BBB) permeability and ultrastructure in brain after diffuse axonal injury (DAI) with secondary brain insults (SBIs). Method: One hundred and twenty Sprague-Dawley (SD) rats were randomised into control, DAI alone and DAI with SBI groups which were sub-divided into 5 groups that were 0.5 h, 2 h, 12 h, 24 h, 48 h post trauma. The animal models of DAI and DAI with SBI have been described before (2). Fluorescence probe Fluo-3/Am was used to measure free Ca2 in neurons. Laser scan microscopy was used to detect fluorescence intensity. After the animals were anesthetized, Lanthanum nitrate liquid was used for intracardiac perfusion to assess BBB permeability. Under the transmission electron microscope, changes in cerebral ultrastructure and BBB permeability were observed. Results: The fluorescence intensity was weak in the control. The concentration of free Ca2 in neurons was obviously increased at 30 min after brain injury, reached a peak at 12 h±24 h (P < 0.01), and appeared to decrease at 48 h after injury. In the DAI alone group, BBB tight junction opening with particles of Lanthanum nitrate outside the vessels was found at 30min after injury, and peaked at 24 h. In DAI with SBI, the changes in ultrastructure and BBB permeability were more severe than that in the DAI alone group at the same time interval. The shape of the fluorescence concentration curve was basically the same for both kinds of brain injury. The intensity of fluorescence in DAI with SBI was higher than that in the DAI alone group at the same time interval (P < 0.05). Conclusion: In DAI alone and DAI with SBI, Ca2 overload and BBB permeability changes interact and both play important roles in the aggravation of brain damage. & 2001 Harcourt Publishers Ltd Keywords: brain, diffuse axonal injury (DAI), secondary brain insult (SBI), calcium, blood±brain barrier (BBB)
INTRODUCTION Brain diffuse axonal injury (DAI) is associated with high morbidity and mortality.1 In a previous study, we successfully established a rat DAI model by lateral rotation of the head, observing the pathological changes of macroscopic petechiae. Additionally at the frontal and temporal bases, axonal retraction balls (ARBs) were observed microscopically, scattered in the brain and brainstem.2 Our previous study on brain thromboxane A2 (TXA2) and prostoglandin I2 (PGI2) levels in diffuse brain injury alone and with hypotension indicated that PGI2 may act as a repair mechanism by providing energy and precursors to the injured tissue and the production of vasoactive arachidonic acid products, especially TXA2, is closely connected to the severity of brain damage.3 Pyrexia, another common symptom seen in multiple-trauma patients with head injury may also worsen the outcome.4 The present study was designed to investigate the changes of neuronal free Ca2 and Received 14 April 2000 Accepted 19 July 2000 Correspondence to: Fei Zhou, Department of Neurosurgery, Xijing Hospital, Xi'an 710032, P. R. China. Tel.: 86 29 2534570; Fax: 86 29 3249273; E-mail: [email protected]
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BBB permeability and ultrastructure in experimental head injury with a pyrexial secondary brain insult (SBI). MATERIAL AND METHODS 1. Animal grouping and model: One hundred and twenty healthy male SD rats, weighing 25020 g, were randomised into control, DAI alone and DAI with SBI groups which were sub-divided into five groups that were 0.5 h, 2 h, 12 h, 24 h, 48 h post trauma. In a previous study, we had successfully produced diffuse axonal injury in rats.2 The same model was used in the present study. All rats were anesthetized with peritoneal injection of 1% sodium pentobarbital (30 mg/kg).The rat's head was horizontally secured to the injury device by two lateral ear bars, a head clip and an anterior teeth hole, with its body 20 oblique to the top of laboratory table. In the injury group, a trigger was pressed and the device instantly rotated the head through a 90 angle in the lateral plane. It had been identified previously that the head rotation was finished in less than 30 milliseconds with an angular velocity above 761 rad/s and an angular acceleration above 1.87 105 rad/s2. In the control group each rat was released from the device once secured. Acute DAI with pyrexia was achieved as Journal of Clinical Neuroscience (2001) 8(6), 557±563