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Changes in blood–brain barrier permeability following neurotoxic lesions of rat brain can be visualised with trypan blue
Journal of Neuroscience Methods 79 (1998) 115 – 121
Changes in blood – brain barrier permeability following neurotoxic lesions of rat brain can be visualised with trypan blue D.S. Reynolds, A.J. Morton * Department of Pharmacology, Uni6ersity of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK Received 18 July 1997; received in revised form 6 October 1997; accepted 8 October 1997
Abstract A simple method for measuring changes in blood–brain barrier (BBB) permeability following neurotoxic lesions is described. In the brains of animals perfused transcardially with a trypan blue solution at the time of sacrifice, the presence of trypan blue staining correlated with changes in BBB function seen with more traditional markers, such as albumin staining. Thus, trypan blue appears to be useful as a marker for changes in BBB permeability. We have used this method to show increases in BBB permeability in striatal lesions induced by three different neurotoxins: chronic systemic injection of 3-nitropropionic acid (3-NP) and intrastriatal injection of either quinolinic or kainic acid. Trypan blue staining was seen in all three types of lesion, with both the neuropil and some neurones being stained. In the kainic acid lesioned animals, trypan blue also stained hippocampal and cortical neurones which are known to degenerate. Our findings suggest that trypan blue makes a more sensitive marker than albumin for both BBB integrity changes and degenerating neurones. Furthermore, this method has the advantages over others of being quick, economic and compatible with most subsequent histological and immunocytochemical staining. © 1998 Elsevier Science B.V. Keywords: Blood–brain barrier; Excitotoxicity; Neurodegeneration; 3-Nitropropionic acid; Rat; Striatum; Trypan blue
1. Introduction The mechanisms of neuronal death following traumatic brain injury or neurotoxic insults are not fully understood but changes in blood – brain barrier (BBB) permeability may play an important role. Increases in BBB permeability have been demonstrated in a number of different types of clinical brain damage, including cerebral ischemia (Olssen et al., 1971; O’Brien et al., 1974; Petito et al., 1982; Nordborg et al., 1991, 1994) and subarachnoid haemorrhage (Lindsberg et al., 1996). Increased permeability of the BBB has also been shown in experimental models of neurodegeneration, including kainic acid-induced seizures (Zucker et al., 1983), intraventricular infusion of N-methyl-D-as* Corresponding author. Tel.: +44 1223 334057; fax: + 44 1223 334040. 0165-0270/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 2 7 0 ( 9 7 ) 0 0 1 6 8 - 4
partate (NMDA; Dietrich et al., 1992), intrastriatal injection of NMDA or endothelin-1 (Miller et al., 1996) and 3-nitropropionic acid (3-NP; Nishino et al., 1995, 1997). Such changes in BBB permeability may contribute to the process of neurodegeneration. Therefore, a simple measure of BBB function would facilitate experimental studies. The integrity of the BBB was first measured using in vivo injection of dyestuffs, such as Evan’s blue and trypan blue (Broman, 1944; Broman et al., 1966). However, these dyes were found to bind plasma proteins (Gregersen and Rawson, 1942; Tschirgi, 1950), and lose their colour intensity during chemical reactions in the tissue. As a result controversy arose as to their reliability for measuring BBB integrity (for review see Dobbing, 1961). These difficulties led to the development of alternative methods, including the in vivo injection of horseradish peroxidase (HRP, Reese and Karnovsky,
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1967; Petito et al., 1982; Broadwell and Sofroniew, 1993), radiolabelled tracers (Zucker et al., 1983; Pont et al., 1995) or fluorescent dyes (Miller et al., 1996). Additionally, immunocytochemistry has been widely used to reveal albumin extravasation (Hamilton and Gould, 1987b; Nordborg et al., 1991; Broadwell and Sofroniew, 1993; Nordborg et al., 1994; Nishino et al., 1995). However, although these methods give satisfactory results, difficulties arise when double labelling studies are attempted. For example, perfusion with HRP is not readily compatible with subsequent processing of the brain for immunocytochemistry because the brain cannot be perfuse-fixed with paraformaldehyde. Furthermore, these methods require in vivo treatments of the animals prior to sacrifice and (particularly for HRP) are expensive. In this study we show that transcardial perfusion of trypan blue at the time of sacrifice makes a reliable marker for BBB permeability changes in rat models of striatal neurodegeneration. There are several advantages of using this system: no in vivo pre-treatment of the animal is required because no peroxidase is involved, fixation with paraformaldehyde is possible and trypan blue does not interfere with subsequent histological and immunocytochemical staining.
2. Methods
2.1. Systemic 3 -NP treatment 3-NP for injection was dissolved in phosphate buffered saline (PBS, 100 mM) and adjusted to pH 7.4 with NaOH, fresh stocks were made up each day. 3-NP was administered by daily subcutaneous injection to rats at an initial dose of 12 mg kg − 1. After every 4 days of treatment, the dose was increased by 3 mg kg − 1 until the rats developed a movement disorder characterised by a wobbly, uncoordinated gait which progressed to hind limb recumbency. At this point 3-NP treatment ended and the rats were allowed to recover (this movement disorder is well correlated with striatal lesion formation (Hamilton and Gould, 1987b; Beal et al., 1993). For treatment 22 female Sprague-Dawley rats (200 –250 g) were used until they developed the characteristic movement disorder. After the cessation of 3-NP injections, rats were allowed to recover for up 4 weeks before being sacrificed. Control animals were treated with PBS alone, instead of 3-NP (n =18).
2.2. Intrastriatal injection of quinolinic or kainic acid Rats were deeply anaesthetised with Avertin (10 ml kg − 1, i.p.) and placed in a stereotaxic frame (Kopf) with the incisor bar 3.3 mm below the interaural line. Using a 10 ml Hamilton syringe, QA (100 nmol) or KA
(3 nmol) was injected into the left striatum, in a 1 ml volume, at a constant rate for a period of 2 min. The coordinates used were anterior 1 mm and lateral 2.5 mm from the bregma and ventral 5 mm from the dura mater (Paxinos and Watson, 1982). After each injection the needle was then left in situ for 2 min before being slowly withdrawn before the incision was closed with interrupted silk sutures. Rats were sacrificed 1 week after lesioning.
2.3. Transcardial perfusion of trypan blue All rats were perfused with a trypan blue solution and then fixed with paraformaldehyde. Trypan blue solution (0.5%) was made by dissolving 1 g of trypan blue in 200 ml PBS, with gentle heat. The solution was allowed to cool to room temperature and then filtered through cotton wool. Heparin (500 units) was added to the filtrate, then the solution was placed on ice and used immediately. The temperature of the trypan blue solution was 10–12°C at the time of perfusion. Rats were anaesthetised with Avertin and perfused transcardially with 200 ml of trypan blue solution, followed by 300 ml of ice-cold paraformaldehyde (2% in PBS). The flow rate of the perfusate was maintained at 25 ml min − 1. The brains were dissected and post-fixed overnight in 2% paraformaldehyde and then cryoprotected in 30% sucrose for 2 days. Subsequently, brains were frozen in powdered dry ice and stored at −80°C until processed for histochemical and immunocytochemical studies. Trypan blue staining was examined in 50 mm thick cryosections cut directly onto gelatincoated slides. Trypan blue staining readily washed out of sections during processing and mounting. To minimise this problem slides were rapidly dehydrated (three washes, 30 s each) in alcohol, defatted in Histolene (Cellpath) and coverslipped with DPX mountant (Agar Scientific). Adjacent sections (30 mm) were cut for immunocytochemistry and histochemistry. Trypan blue staining washed out of the sections during subsequent staining and hence did not interfere with these processes.
2.4. Albumin immunocytochemistry Free-floating cryostat sections were stained for albumin immunoreactivity. Non-specific binding was blocked by incubating the sections at 4°C overnight in blocking solution (3% normal deer serum in PBS containing 0.2% Triton X-100) with 0.02% sodium azide added. Sections were then incubated in sheep polyclonal albumin antisera (Serotec) diluted to a concentration of 1:1000 in blocking solution for 1 week at 4°C. Then sections were washed five times; for 5 min each; in wash solution (PBS containing 0.02% Triton X-100) and then incubated at 4°C overnight in HRP-la-
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Fig. 1. Parallel coronal brain sections at the level of the striatum from a rat treated with either saline (a, c and e) or 3-NP (b, d and f). The cortex (ctx), corpus callosum (cc) and striatum (st) have been indicated. Transcardial perfusion with trypan blue 1 week after the end of treatment revealed very little staining in the saline-treated rat brain (a). In contrast, the 3-NP-treated brain displayed bilateral striatal lesions (arrows, b). The increase in trypan blue staining correlated with an increase in albumin immunoreactivity in a parallel section (arrows, d). The area marked by trypan blue and albumin staining corresponded to the lesion area, as shown by a decrease in NADPH diaphorase activity (arrows, f). There was no specific albumin staining in control brain sections (c), nor did NADPH diaphorase staining reveal lesions. Bar =2 mm.
belled rabbit anti-sheep secondary antibody (Sigma) diluted to 1:1000 in blocking solution without azide. The sections were then washed five times, for 5 min with wash solution and developed with 3,3%-diaminobenzidine (0.5 mg ml − 1) in 50 mM Tris buffer (pH 7.6), containing 0.009% hydrogen peroxide.
2.5. NADPH diaphorase and cresyl 6iolet histochemistry The presence and position of lesions was determined using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and cresyl violet histochemical staining. NADPH diaphorase staining was performed on free-floating cryostat sections by incubating them for 60 min in the dark, at 37°C in a reaction mixture containing 50 mM PBS, 5 mM MgCl2, 2 mg ml − 1 NADPH (reduced form) and 1 mg ml − 1 nitro blue tetrazolium.
Sections for cresyl violet staining were mounted onto gelatin-coated slides, dehydrated through graded alcohol and defatted in Histolene for 20 min. They were then incubated in a 1% solution of cresyl violet acetate for 30 min, differentiated in 1% acetic acid in 70% aqueous alcohol, before being dehydrated and mounted in DPX.
3. Results and discussion In saline-treated control rat brain sections (Fig. 1(a)) there was no trypan blue staining visible in any region, except in the circumventricular organs which lie outside the BBB (Fig. 2). The brain regions displayed intense cellular staining, as well as paler neuropil staining which had partially diffused into adjacent areas. Parallel sections stained for albumin revealed similar staining
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Fig. 2. Both trypan blue (a and c) and albumin immunocytochemistry (b and d) stained the circumventricular organs, which lie outside the BBB. The subfornical organ (SFO) displayed strong trypan blue cellular staining (a), with some of the dye diffusing into the neighbouring ventral hippocampal commissure (vhc). The choroid plexus (ChP) also stained strongly with trypan blue. Similarly, albumin immunoreactivity was seen in the SFO and ChP (c). The arcuate nucleus (Arc) and median eminence (ME) stained intensely for both trypan blue (b), and albumin (d). D3V, dorsal third ventricle; 3V, third ventricle. Bar = 500 mm.
only in the circumventricular organs (Fig. 1(c) Fig. 2). Animals treated with 3-NP displayed bilateral striatal lesions, whereas those treated with either quinolinic or kainic acid only had lesions in the striatum on the side in which the toxin was injected (Fig. 1(d) Fig. 3). Throughout the lesions cresyl violet staining showed a marked loss of neuronal cell bodies and an increase in glial cells (Coyle and Schwarcz, 1976; Beal et al., 1986, 1993). NADPH diaphorase activity was markedly decreased within the lesion area and the boundary of the lesion was well delineated by this stain (Fig. 1(f), Fig. 3(c d)). There was intense bilateral trypan blue staining within the striatal lesions of 3-NP-treated rats (Fig. 1(b)) and unilateral staining in those treated with both quinolinic or kainic acid (Fig. 3(a b)). Parallel sections from these animals also showed intense albumin staining throughout the lesions (Fig. 1(d)), suggesting that trypan blue was a good marker of changes in permeability of the BBB. Damage to the integrity of the BBB occurred rapidly after 3-NP-induced lesion formation. The earliest time point we examined was 2 h after the onset of clinical symptoms. In these rats there was a marked increase in trypan blue staining of both the neuropil and cell
bodies within the striatal lesions (data not shown). This increased permeability to trypan blue persisting for at least 4 weeks, although the amount of staining decreased between 1 and 4 weeks after lesion formation. Similar results have been found previously, using albumin immunocytochemistry (Hamilton and Gould, 1987a) These workers found that at 1 h after the onset of clinical symptoms of lesion development there was no gross albumin immunoreactivity, although there was specific staining surrounding the capillaries within the lesion area. After 2.5 h there was strong immunoreactivity throughout the lesion area at a gross level. Nishino and colleagues have also shown that BBB permeability is maintained for several weeks following 3-NP treatment (Nishino et al., 1995, 1997). Hence, the increase in trypan blue staining seen during striatal lesion formation in our study is likely to be due to an increase in BBB permeability. In addition to the clear delineation of the lesion area at a macroscopic level, trypan blue staining revealed interesting details at a microscopic level. Within the striatal lesions induced by both chronic and acute treatments (Fig. 4(b c)), trypan blue strongly stained the neuropil and cell bodies, but not the fibre bundles. The
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Fig. 3. Coronal rats brain sections from rats that received intrastriatal injections of either quinolinic acid (a, c and e) or kainic acid (b, d and f). The trypan blue staining (a and b) was intense staining throughout the injected striatum (left side) after both treatments. Following quinolinic acid injection, the neurodegeneration is restricted to the striatum, as shown by a loss of NADPH diaphorase staining (c). However, kainic acid injection causes neuronal loss from extrastriatal areas, including the deep layers of the overlying cortex (arrowheads, d), and the CA3 cells of the ipsilateral hippocampus (arrowheads, f). Trypan blue marks these dying neurones. Extrastriatal damage is not a feature of quinolinic acid injection and no hippocampal neurones stain for trypan blue (e), although a few cortical neurones are marked. L, lesion; Hf, hippocampus; ME, median eminence. Bar =2 mm.
neuropil staining was heterogeneous, with darker areas of staining distributed predominantly around the edges of the lesions. Intensely stained cells were visible within the dye-stained neuropil, with the number, size and morphology of cells suggesting that they are neurones. Since neurones marked by trypan blue are those within the area that degenerates in these models, it seems likely that they are damaged. One can therefore speculate that the dye should mark other neurones that are susceptible to degeneration. One such group of neurones is the CA3 cells of the hippocampus, which degenerate following kainic acid injection (Wuerthele et al., 1978; Schwob et al., 1980; Zaczek et al., 1980). To test this hypothesis we examined the CA3 hippocampal neurones and the deep-layer cortical neurones in the brains of rats that had received intrastriatal kainic acid
lesions. Following kainic acid injection, the ipsilateral CA3 cells of the hippocampus stained positively for trypan blue, whereas the contralateral hippocampal neurones were unstained (Fig. 3(f) Fig. 4(d)). Furthermore, the deep layer cortical neurones overlying the damaged striatum were also dye-positive, but the contralateral cortical neurones were unstained (Fig. 3(b)).This is consistent with the site of distal damage reported by other groups in the hippocampus and cortex (Wuerthele et al., 1978; Schwob et al., 1980; Zaczek et al., 1980). To test that this staining was not just due to damage to the neurones during the perfusion procedure, the hippocampus of quinolinic acid lesioned rats was also examined. Extrastriatal damage is not a common feature of intrastriatal quinolinic acid injection and accordingly there was no trypan blue
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Fig. 4. Perfusion of trypan blue produced precipitate artefacts in blood vessels (a). These were clearly non-cellular and could be easily distinguished from specific labelling of degenerating neurones. Within both the 3-NP (b) and kainic acid-induced (c) striatal lesions there was heterogeneous neuropil staining with the fibre bundles remaining unstained (asterisks). Intensely stained cells could be seen within the dye-positive neuropil; their number, size and morphology suggested that they are neurones. Following intrastriatal injection of kainic acid, the ipsilateral CA3 hippocampal cells degenerate and these neurones stain for trypan blue (d). Bar =100 mm.
labelling of hippocampal neurones in any of the quinolinic acid lesioned brains (although a few cortical neurones were stained, particularly in the piriform cortex (Fig. 3(e))). Trypan blue appears to be a sensitive marker of neurodegeneration. In fact it may be a better marker for changes in BBB integrity in distal regions than albumin staining, since it is very difficult to visualise hippocampal damage using albumin immunocytochemistry. Although occasional artefacts were seen following transcardial perfusion with trypan blue, these were very different and easily distinguished from the trypan blue staining associated with lesions. The most common artefactual staining appears to be largely due to precipitates of trypan blue becoming lodged in small blood vessels (Fig. 4(a)). An increased number of such inclusions were found in 3-NP-treated rat brains, compared to saline-treated brains. Nishino et al. (1995) noted that after prolonged 3-NP treatment, rats that did not develop striatal lesions displayed immunoreactivity for vascular elements around striatal blood vessels, suggesting that a mild dysfunction of the BBB had already
occurred. Mild damage to striatal blood vessels may explain the increased number of trypan blue precipitates in 3-NP intoxicated rat brains. Similar artefacts were found in the study of Broadwell and Sofroniew (1993) using albumin extravasation, but these were also clearly distinguishable from staining of BBB breakdown. With our method precipitate inclusions could be minimised by ensuring that the trypan blue solution cooled slowly and was used immediately after filtration. Our data suggest that transcardial perfusion of rats with a trypan blue solution at the time of sacrifice provides a reliable marker of BBB permeability. Trypan blue stains only regions outside the BBB in control rats, but stains neuropil as well as cells within the area of striatal damage. In addition, in the kainic acid model, distal regions that undergo degeneration (such as the hippocampus) are also stained. The trypan blue method is an improvement over existing methods for examining BBB permeability, because it is sensitive and can be performed using standard perfusion techniques. Moreover, trypan blue staining does not interfere with most subsequent histochemical or immunocytochemical pro-
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cedures, thus making it the ideal marker for BBB permeability studies.
Acknowledgements This work was supported by grants from the Wellcome Trust and the Hereditary Disease Foundation. We thank Mr Roger Hart for excellent photography and Mrs Wendy Leavens for expert technical assistance.
References Beal MF, Kowall NW, Ellison DW, Mazurek MF, Swartz KJ, Martin JB. Replication of the neurochemical characteristics of Huntington’s disease by quinolinic acid. Nature 1986;321:168 – 71. Beal MF, Brouillet E, Jenkins BG, et al. Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J Neurosci 1993;13:4181 – 92. Broadwell RD, Sofroniew MV. Serum proteins bypass the blood – brain fluid barriers for extracellular entry to the central nervous system. Exp Neurol 1993;120:245–63. Broman T. Supravital analysis of disorders in the cerebral vascular permeability in man. Acta Medica Scand 1944;118:79–83. Broman T, Branemark P-I, Johansson B, Steinwall O. Intravital and postmortem studies on air embolism damage of the blood – brain barrier tested with trypan blue. Acta Neurol Scand 1966;42:146 – 52. Coyle JT, Schwarcz R. Lesion of striatal neurones with kainic acid provides a model of Huntington’s chorea. Nature 1976;263:244 – 6. Dietrich WD, Alonso O, Halley M, Busto R, Globus MY-T. Intraventricular infusion of N-methyl-D-aspartate. 1. Acute blood – brain barrier consequences. Acta Neuropathol 1992;84:621 – 9. Dobbing J. The blood – brain barrier. Physiological Rev 1961;41:135 – 89. Gregersen MI, Rawson RA. The disappearance of T-1824 and structurally related dyes from the blood stream. Am J Physiol 1942;138:698 – 707. Hamilton BF, Gould DH. Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage. Acta Neuropathol 1987a;72:286 – 97. Hamilton BF, Gould DH. Correlation of morphologic brain lesions with physiologic alterations and blood–brain barrier impairment in 3-nitropropionic acid toxicity in rats. Acta Neuropathol 1987b;74:67 – 74.
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Lindsberg PJ, Ohman J, Lehto T, et al. Complement activation in the central nervous system following blood – brain barrier damage in man. Ann Neurol 1996;40:587 – 96. Miller D, Monsul NT, Vender JR, Lehmann JC. NMDA- and endothelin-1-induced increases in blood – brain barrier permeability quantitated with Lucifer yellow. J Neurol Sci 1996;136:37–40. Nishino H, Shimano Y, Kumazaki M, Sakurai T. Chronically administered 3-nitropropionic acid induces striatal lesions attributed to dysfunction of the blood – brain barrier. Neurosci Lett 1995;186:161 – 4. Nishino H, Kumazaki M, Fukuda A, et al. Acute 3-nitropropionic acid intoxication induces striatal astrocytic cell death and dysfunction of the blood – brain barrier: involvement of dopamine toxicity. Neurosci Res 1997;27:343 – 55. Nordborg C, Sokrab TEO, Johansson BB. The relationship between plasma protein extravasation and remote tissue changes after experimental brain infarction. Acta Neuropathol 1991;82:118–26. Nordborg C, Sokrab TEO, Johansson BB. Oedema-related tissue damage after temporary and permanent occlusion of the middle cerebral artery. Neuropath Appl Neurobiol 1994;20:56 –65. O’Brien MD, Jordan MM, Waltz AG. Ischemic cerebral edema and the blood – brain barrier: distributions of pertechnetate, albumin, sodium and antipyrine in brains of cats after occlusion of the midde cerebral artery. Arch Neurol 1974;18:461 – 5. Olssen Y, Crowell RM, Klatzo I. Permeability of the blood–brain barrier to protein tracers in focal ischemia and infarction caused by occlusion of the middle cerebral artery. Acta Neuropathol 1971;18:89 – 102. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press, 1982. Petito CK, Pulsinelli WA, Jacobsen G, Plum F. Edema and vascular permeability in cerebral ischemia: comparison between ischemic neuronal damage and infarction. J Neuropathol Exp Neurol 1982;41:423 – 36. Pont F, Collet A, Lallement G. Early and transient increase of rat hippocampal blood – brain barrier permeability to amino acids during kainic acid-induced seizures. Neurosci Lett 1995;184:52–4. Reese TS, Karnovsky MJ. Fine structural localization of a blood– brain barrier to exogenous peroxidase. J Cell Biol 1967;34:207– 17. Schwob JE, Fuller T, Price JL, Olney JW. Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histologial study. Neuroscience 1980;5:991–1014. Tschirgi RD. Protein complexes and the impermeability of the blood – brain barrier to dyes. Am J Physiol 1950;163:756. Wuerthele SM, Lovell KJ, Jones MZ, Moore KE. A histological study of kainic acid-induced lesions in the rat brain. Brain Res 1978;149:489 – 97. Zaczek R, Simonton S, Coyle JT. Local and distant neuronal degeneration following intrastriatal injection of kainic acid. J Neuropathol Exp Neurol 1980;39:245 – 64. Zucker DK, Wooten GF, Lothman EW. Blood – brain barrier changes with kainic acid-induced limbic seizures. Exp Neurol 1983;79:422 – 33.
Changes in blood – brain barrier permeability following neurotoxic lesions of rat brain can be visualised with trypan blue D.S. Reynolds, A.J. Morton * Department of Pharmacology, Uni6ersity of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK Received 18 July 1997; received in revised form 6 October 1997; accepted 8 October 1997
Abstract A simple method for measuring changes in blood–brain barrier (BBB) permeability following neurotoxic lesions is described. In the brains of animals perfused transcardially with a trypan blue solution at the time of sacrifice, the presence of trypan blue staining correlated with changes in BBB function seen with more traditional markers, such as albumin staining. Thus, trypan blue appears to be useful as a marker for changes in BBB permeability. We have used this method to show increases in BBB permeability in striatal lesions induced by three different neurotoxins: chronic systemic injection of 3-nitropropionic acid (3-NP) and intrastriatal injection of either quinolinic or kainic acid. Trypan blue staining was seen in all three types of lesion, with both the neuropil and some neurones being stained. In the kainic acid lesioned animals, trypan blue also stained hippocampal and cortical neurones which are known to degenerate. Our findings suggest that trypan blue makes a more sensitive marker than albumin for both BBB integrity changes and degenerating neurones. Furthermore, this method has the advantages over others of being quick, economic and compatible with most subsequent histological and immunocytochemical staining. © 1998 Elsevier Science B.V. Keywords: Blood–brain barrier; Excitotoxicity; Neurodegeneration; 3-Nitropropionic acid; Rat; Striatum; Trypan blue
1. Introduction The mechanisms of neuronal death following traumatic brain injury or neurotoxic insults are not fully understood but changes in blood – brain barrier (BBB) permeability may play an important role. Increases in BBB permeability have been demonstrated in a number of different types of clinical brain damage, including cerebral ischemia (Olssen et al., 1971; O’Brien et al., 1974; Petito et al., 1982; Nordborg et al., 1991, 1994) and subarachnoid haemorrhage (Lindsberg et al., 1996). Increased permeability of the BBB has also been shown in experimental models of neurodegeneration, including kainic acid-induced seizures (Zucker et al., 1983), intraventricular infusion of N-methyl-D-as* Corresponding author. Tel.: +44 1223 334057; fax: + 44 1223 334040. 0165-0270/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 2 7 0 ( 9 7 ) 0 0 1 6 8 - 4
partate (NMDA; Dietrich et al., 1992), intrastriatal injection of NMDA or endothelin-1 (Miller et al., 1996) and 3-nitropropionic acid (3-NP; Nishino et al., 1995, 1997). Such changes in BBB permeability may contribute to the process of neurodegeneration. Therefore, a simple measure of BBB function would facilitate experimental studies. The integrity of the BBB was first measured using in vivo injection of dyestuffs, such as Evan’s blue and trypan blue (Broman, 1944; Broman et al., 1966). However, these dyes were found to bind plasma proteins (Gregersen and Rawson, 1942; Tschirgi, 1950), and lose their colour intensity during chemical reactions in the tissue. As a result controversy arose as to their reliability for measuring BBB integrity (for review see Dobbing, 1961). These difficulties led to the development of alternative methods, including the in vivo injection of horseradish peroxidase (HRP, Reese and Karnovsky,
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1967; Petito et al., 1982; Broadwell and Sofroniew, 1993), radiolabelled tracers (Zucker et al., 1983; Pont et al., 1995) or fluorescent dyes (Miller et al., 1996). Additionally, immunocytochemistry has been widely used to reveal albumin extravasation (Hamilton and Gould, 1987b; Nordborg et al., 1991; Broadwell and Sofroniew, 1993; Nordborg et al., 1994; Nishino et al., 1995). However, although these methods give satisfactory results, difficulties arise when double labelling studies are attempted. For example, perfusion with HRP is not readily compatible with subsequent processing of the brain for immunocytochemistry because the brain cannot be perfuse-fixed with paraformaldehyde. Furthermore, these methods require in vivo treatments of the animals prior to sacrifice and (particularly for HRP) are expensive. In this study we show that transcardial perfusion of trypan blue at the time of sacrifice makes a reliable marker for BBB permeability changes in rat models of striatal neurodegeneration. There are several advantages of using this system: no in vivo pre-treatment of the animal is required because no peroxidase is involved, fixation with paraformaldehyde is possible and trypan blue does not interfere with subsequent histological and immunocytochemical staining.
2. Methods
2.1. Systemic 3 -NP treatment 3-NP for injection was dissolved in phosphate buffered saline (PBS, 100 mM) and adjusted to pH 7.4 with NaOH, fresh stocks were made up each day. 3-NP was administered by daily subcutaneous injection to rats at an initial dose of 12 mg kg − 1. After every 4 days of treatment, the dose was increased by 3 mg kg − 1 until the rats developed a movement disorder characterised by a wobbly, uncoordinated gait which progressed to hind limb recumbency. At this point 3-NP treatment ended and the rats were allowed to recover (this movement disorder is well correlated with striatal lesion formation (Hamilton and Gould, 1987b; Beal et al., 1993). For treatment 22 female Sprague-Dawley rats (200 –250 g) were used until they developed the characteristic movement disorder. After the cessation of 3-NP injections, rats were allowed to recover for up 4 weeks before being sacrificed. Control animals were treated with PBS alone, instead of 3-NP (n =18).
2.2. Intrastriatal injection of quinolinic or kainic acid Rats were deeply anaesthetised with Avertin (10 ml kg − 1, i.p.) and placed in a stereotaxic frame (Kopf) with the incisor bar 3.3 mm below the interaural line. Using a 10 ml Hamilton syringe, QA (100 nmol) or KA
(3 nmol) was injected into the left striatum, in a 1 ml volume, at a constant rate for a period of 2 min. The coordinates used were anterior 1 mm and lateral 2.5 mm from the bregma and ventral 5 mm from the dura mater (Paxinos and Watson, 1982). After each injection the needle was then left in situ for 2 min before being slowly withdrawn before the incision was closed with interrupted silk sutures. Rats were sacrificed 1 week after lesioning.
2.3. Transcardial perfusion of trypan blue All rats were perfused with a trypan blue solution and then fixed with paraformaldehyde. Trypan blue solution (0.5%) was made by dissolving 1 g of trypan blue in 200 ml PBS, with gentle heat. The solution was allowed to cool to room temperature and then filtered through cotton wool. Heparin (500 units) was added to the filtrate, then the solution was placed on ice and used immediately. The temperature of the trypan blue solution was 10–12°C at the time of perfusion. Rats were anaesthetised with Avertin and perfused transcardially with 200 ml of trypan blue solution, followed by 300 ml of ice-cold paraformaldehyde (2% in PBS). The flow rate of the perfusate was maintained at 25 ml min − 1. The brains were dissected and post-fixed overnight in 2% paraformaldehyde and then cryoprotected in 30% sucrose for 2 days. Subsequently, brains were frozen in powdered dry ice and stored at −80°C until processed for histochemical and immunocytochemical studies. Trypan blue staining was examined in 50 mm thick cryosections cut directly onto gelatincoated slides. Trypan blue staining readily washed out of sections during processing and mounting. To minimise this problem slides were rapidly dehydrated (three washes, 30 s each) in alcohol, defatted in Histolene (Cellpath) and coverslipped with DPX mountant (Agar Scientific). Adjacent sections (30 mm) were cut for immunocytochemistry and histochemistry. Trypan blue staining washed out of the sections during subsequent staining and hence did not interfere with these processes.
2.4. Albumin immunocytochemistry Free-floating cryostat sections were stained for albumin immunoreactivity. Non-specific binding was blocked by incubating the sections at 4°C overnight in blocking solution (3% normal deer serum in PBS containing 0.2% Triton X-100) with 0.02% sodium azide added. Sections were then incubated in sheep polyclonal albumin antisera (Serotec) diluted to a concentration of 1:1000 in blocking solution for 1 week at 4°C. Then sections were washed five times; for 5 min each; in wash solution (PBS containing 0.02% Triton X-100) and then incubated at 4°C overnight in HRP-la-
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Fig. 1. Parallel coronal brain sections at the level of the striatum from a rat treated with either saline (a, c and e) or 3-NP (b, d and f). The cortex (ctx), corpus callosum (cc) and striatum (st) have been indicated. Transcardial perfusion with trypan blue 1 week after the end of treatment revealed very little staining in the saline-treated rat brain (a). In contrast, the 3-NP-treated brain displayed bilateral striatal lesions (arrows, b). The increase in trypan blue staining correlated with an increase in albumin immunoreactivity in a parallel section (arrows, d). The area marked by trypan blue and albumin staining corresponded to the lesion area, as shown by a decrease in NADPH diaphorase activity (arrows, f). There was no specific albumin staining in control brain sections (c), nor did NADPH diaphorase staining reveal lesions. Bar =2 mm.
belled rabbit anti-sheep secondary antibody (Sigma) diluted to 1:1000 in blocking solution without azide. The sections were then washed five times, for 5 min with wash solution and developed with 3,3%-diaminobenzidine (0.5 mg ml − 1) in 50 mM Tris buffer (pH 7.6), containing 0.009% hydrogen peroxide.
2.5. NADPH diaphorase and cresyl 6iolet histochemistry The presence and position of lesions was determined using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and cresyl violet histochemical staining. NADPH diaphorase staining was performed on free-floating cryostat sections by incubating them for 60 min in the dark, at 37°C in a reaction mixture containing 50 mM PBS, 5 mM MgCl2, 2 mg ml − 1 NADPH (reduced form) and 1 mg ml − 1 nitro blue tetrazolium.
Sections for cresyl violet staining were mounted onto gelatin-coated slides, dehydrated through graded alcohol and defatted in Histolene for 20 min. They were then incubated in a 1% solution of cresyl violet acetate for 30 min, differentiated in 1% acetic acid in 70% aqueous alcohol, before being dehydrated and mounted in DPX.
3. Results and discussion In saline-treated control rat brain sections (Fig. 1(a)) there was no trypan blue staining visible in any region, except in the circumventricular organs which lie outside the BBB (Fig. 2). The brain regions displayed intense cellular staining, as well as paler neuropil staining which had partially diffused into adjacent areas. Parallel sections stained for albumin revealed similar staining
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Fig. 2. Both trypan blue (a and c) and albumin immunocytochemistry (b and d) stained the circumventricular organs, which lie outside the BBB. The subfornical organ (SFO) displayed strong trypan blue cellular staining (a), with some of the dye diffusing into the neighbouring ventral hippocampal commissure (vhc). The choroid plexus (ChP) also stained strongly with trypan blue. Similarly, albumin immunoreactivity was seen in the SFO and ChP (c). The arcuate nucleus (Arc) and median eminence (ME) stained intensely for both trypan blue (b), and albumin (d). D3V, dorsal third ventricle; 3V, third ventricle. Bar = 500 mm.
only in the circumventricular organs (Fig. 1(c) Fig. 2). Animals treated with 3-NP displayed bilateral striatal lesions, whereas those treated with either quinolinic or kainic acid only had lesions in the striatum on the side in which the toxin was injected (Fig. 1(d) Fig. 3). Throughout the lesions cresyl violet staining showed a marked loss of neuronal cell bodies and an increase in glial cells (Coyle and Schwarcz, 1976; Beal et al., 1986, 1993). NADPH diaphorase activity was markedly decreased within the lesion area and the boundary of the lesion was well delineated by this stain (Fig. 1(f), Fig. 3(c d)). There was intense bilateral trypan blue staining within the striatal lesions of 3-NP-treated rats (Fig. 1(b)) and unilateral staining in those treated with both quinolinic or kainic acid (Fig. 3(a b)). Parallel sections from these animals also showed intense albumin staining throughout the lesions (Fig. 1(d)), suggesting that trypan blue was a good marker of changes in permeability of the BBB. Damage to the integrity of the BBB occurred rapidly after 3-NP-induced lesion formation. The earliest time point we examined was 2 h after the onset of clinical symptoms. In these rats there was a marked increase in trypan blue staining of both the neuropil and cell
bodies within the striatal lesions (data not shown). This increased permeability to trypan blue persisting for at least 4 weeks, although the amount of staining decreased between 1 and 4 weeks after lesion formation. Similar results have been found previously, using albumin immunocytochemistry (Hamilton and Gould, 1987a) These workers found that at 1 h after the onset of clinical symptoms of lesion development there was no gross albumin immunoreactivity, although there was specific staining surrounding the capillaries within the lesion area. After 2.5 h there was strong immunoreactivity throughout the lesion area at a gross level. Nishino and colleagues have also shown that BBB permeability is maintained for several weeks following 3-NP treatment (Nishino et al., 1995, 1997). Hence, the increase in trypan blue staining seen during striatal lesion formation in our study is likely to be due to an increase in BBB permeability. In addition to the clear delineation of the lesion area at a macroscopic level, trypan blue staining revealed interesting details at a microscopic level. Within the striatal lesions induced by both chronic and acute treatments (Fig. 4(b c)), trypan blue strongly stained the neuropil and cell bodies, but not the fibre bundles. The
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Fig. 3. Coronal rats brain sections from rats that received intrastriatal injections of either quinolinic acid (a, c and e) or kainic acid (b, d and f). The trypan blue staining (a and b) was intense staining throughout the injected striatum (left side) after both treatments. Following quinolinic acid injection, the neurodegeneration is restricted to the striatum, as shown by a loss of NADPH diaphorase staining (c). However, kainic acid injection causes neuronal loss from extrastriatal areas, including the deep layers of the overlying cortex (arrowheads, d), and the CA3 cells of the ipsilateral hippocampus (arrowheads, f). Trypan blue marks these dying neurones. Extrastriatal damage is not a feature of quinolinic acid injection and no hippocampal neurones stain for trypan blue (e), although a few cortical neurones are marked. L, lesion; Hf, hippocampus; ME, median eminence. Bar =2 mm.
neuropil staining was heterogeneous, with darker areas of staining distributed predominantly around the edges of the lesions. Intensely stained cells were visible within the dye-stained neuropil, with the number, size and morphology of cells suggesting that they are neurones. Since neurones marked by trypan blue are those within the area that degenerates in these models, it seems likely that they are damaged. One can therefore speculate that the dye should mark other neurones that are susceptible to degeneration. One such group of neurones is the CA3 cells of the hippocampus, which degenerate following kainic acid injection (Wuerthele et al., 1978; Schwob et al., 1980; Zaczek et al., 1980). To test this hypothesis we examined the CA3 hippocampal neurones and the deep-layer cortical neurones in the brains of rats that had received intrastriatal kainic acid
lesions. Following kainic acid injection, the ipsilateral CA3 cells of the hippocampus stained positively for trypan blue, whereas the contralateral hippocampal neurones were unstained (Fig. 3(f) Fig. 4(d)). Furthermore, the deep layer cortical neurones overlying the damaged striatum were also dye-positive, but the contralateral cortical neurones were unstained (Fig. 3(b)).This is consistent with the site of distal damage reported by other groups in the hippocampus and cortex (Wuerthele et al., 1978; Schwob et al., 1980; Zaczek et al., 1980). To test that this staining was not just due to damage to the neurones during the perfusion procedure, the hippocampus of quinolinic acid lesioned rats was also examined. Extrastriatal damage is not a common feature of intrastriatal quinolinic acid injection and accordingly there was no trypan blue
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Fig. 4. Perfusion of trypan blue produced precipitate artefacts in blood vessels (a). These were clearly non-cellular and could be easily distinguished from specific labelling of degenerating neurones. Within both the 3-NP (b) and kainic acid-induced (c) striatal lesions there was heterogeneous neuropil staining with the fibre bundles remaining unstained (asterisks). Intensely stained cells could be seen within the dye-positive neuropil; their number, size and morphology suggested that they are neurones. Following intrastriatal injection of kainic acid, the ipsilateral CA3 hippocampal cells degenerate and these neurones stain for trypan blue (d). Bar =100 mm.
labelling of hippocampal neurones in any of the quinolinic acid lesioned brains (although a few cortical neurones were stained, particularly in the piriform cortex (Fig. 3(e))). Trypan blue appears to be a sensitive marker of neurodegeneration. In fact it may be a better marker for changes in BBB integrity in distal regions than albumin staining, since it is very difficult to visualise hippocampal damage using albumin immunocytochemistry. Although occasional artefacts were seen following transcardial perfusion with trypan blue, these were very different and easily distinguished from the trypan blue staining associated with lesions. The most common artefactual staining appears to be largely due to precipitates of trypan blue becoming lodged in small blood vessels (Fig. 4(a)). An increased number of such inclusions were found in 3-NP-treated rat brains, compared to saline-treated brains. Nishino et al. (1995) noted that after prolonged 3-NP treatment, rats that did not develop striatal lesions displayed immunoreactivity for vascular elements around striatal blood vessels, suggesting that a mild dysfunction of the BBB had already
occurred. Mild damage to striatal blood vessels may explain the increased number of trypan blue precipitates in 3-NP intoxicated rat brains. Similar artefacts were found in the study of Broadwell and Sofroniew (1993) using albumin extravasation, but these were also clearly distinguishable from staining of BBB breakdown. With our method precipitate inclusions could be minimised by ensuring that the trypan blue solution cooled slowly and was used immediately after filtration. Our data suggest that transcardial perfusion of rats with a trypan blue solution at the time of sacrifice provides a reliable marker of BBB permeability. Trypan blue stains only regions outside the BBB in control rats, but stains neuropil as well as cells within the area of striatal damage. In addition, in the kainic acid model, distal regions that undergo degeneration (such as the hippocampus) are also stained. The trypan blue method is an improvement over existing methods for examining BBB permeability, because it is sensitive and can be performed using standard perfusion techniques. Moreover, trypan blue staining does not interfere with most subsequent histochemical or immunocytochemical pro-
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cedures, thus making it the ideal marker for BBB permeability studies.
Acknowledgements This work was supported by grants from the Wellcome Trust and the Hereditary Disease Foundation. We thank Mr Roger Hart for excellent photography and Mrs Wendy Leavens for expert technical assistance.
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