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Cholinergic innervation of pial arteries in senescent rats: an immunohistochemical study
Mechanisms of Ageing and Development 123 (2002) 529– 536 www.elsevier.com/locate/mechagedev
Cholinergic innervation of pial arteries in senescent rats: an immunohistochemical study Dahuk El-Assouad *, Seyed Khosrow Tayebati Sezione di Anatomia Umana, Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Uni6ersita` di Camerino, Via Scalzino, 3, 62032 Camerino, Italy Received 16 February 2001; received in revised form 11 June 2001; accepted 27 July 2001
Abstract Perivascular acetylcholine (ACh)-immunoreactive nerve fibres were demonstrated in basilar and middle cerebral arteries, in pial arteries and arterioles and in intracerebral arteries of male Fisher 344 rats of 6 months (young), 15 months (adult) and 22 months (senescent). Analysis included whole mounts of basilar and middle cerebral arteries, of pial arteries and sections of brain including pia-arachnoid membrane to demonstrate the localization of nerve fibres throughout the wall of pial and of intracerebral arteries. ACh-immunoreactive nerve fibres were demonstrated by indirect immunohistochemistry using a polyclonal anti-ACh antibody and their relative density was quantified. Perivascular ACh-immunoreactive nerve fibres were located in basilar and middle cerebral arteries, in pial arteries and arterioles and in intracerebral arteries. These fibres were found in the adventitia and adventitia– media border with a higher density in pial rather than in intracerebral arteries. A decrease of ACh-immunoreactive nerve fibres was observed both in pial and intracerebral arteries of adult or senescent rats compared to younger cohorts. The direct demonstration of ACh-immunoreactive nerve fibres in the cerebrovascular tree may contribute to evaluate the influence of experimental and pathological conditions on cerebrovascular cholinergic neuroeffector mechanisms, including a role of cholinergic innervation in the pathophysiology of cerebrovascular disease of the elderly. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral arteries; Acetylcholine; Immunohistochemistry; Aging; Fisher 344 rats
1. Introduction The existence of an autonomic nervous control of cerebral vessels was extensively documented by
The present work was awarded the young neuromorphologist G.I.S.N. prize for 2000 to Dr. D. El-Assouad. * Corresponding author. Tel.: +39-0737-403311; fax: + 390737-630618. E-mail address: [email protected] (D. El-Assouad).
histochemical, ultrastructural, and pharmacological studies. The best known autonomic supply of cerebrovascular tree is that provided by sympathetic nervous system. Large and small cerebral arteries, pial and parenchymal arteries, and veins receive a dense sympathetic innervation (Nielsen and Owman, 1967; Purdy and Bevan, 1977), the density of which in different brain areas is rather heterogeneous (Edvinsson, 1975b). Sympathetic neuroeffector junctions release noradrenaline.
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This neurotransmitter has a primarily vasoconstrictor function in the cerebrovascular tree (Kuschinsky and Wahl, 1975; Wei et al., 1975). Sympathetic nerves to cerebral vessels can contain also neuropeptide Y acting as a co-transmitter with noradrenaline. The neurogenic vasoconstriction induced by activity of this subpopulation of nerve fibres is mainly due to the release of neuropeptide Y, whereas noradrenaline or other vasoconstrictor agents do not contribute significantly to the increase of cerebrovascular tone. Cerebrovascular sympathetic nerves containing neuropeptide Y are probably active in cerebral auto-regulation (Branston, 1995), although sympathetic innervation of cerebral vessels influences cerebral blood flow (CBF) mainly under conditions of cerebrovascular stress rather than in normal conditions (Edvinsson et al., 1993; Branston, 1995). The presence of non-sympathetic cholinergic nerve fibres in the brain vasculature has been a matter of controversy. In fact, microanatomical demonstration of parasympathetic nerves in the cerebral blood vessels has been hindered by the lack of suitable markers for the identification of cholinergic nerves. The first studies on the topic used the acetylcholine (ACh) catabolic enzyme acetylcholinesterase (AChE) as a marker of cholinergic nerves. Unfortunately, also some populations of sympathetic nerves express AChE and the enzyme degrades not only ACh, but also some neuropeptides present in autonomic nerves (Amenta et al., 1980b). Hence, AChE cannot be considered as a reliable marker of cholinergic non-sympathetic nerves (Amenta et al., 1980b). Perivascular AChE-positive nerve fibres were visualized in cerebral blood vessels of several species including humans (Edvinsson et al., 1976), monkey (Denn and Stone, 1976), dog (Amenta et al., 1980a), rabbit (Edvinsson et al., 1972; Baramidze et al., 1982), rat (Edvinsson et al., 1972; Licata et al., 1975; Vasquez and Purves, 1979; Kobayashi et al., 1983; Hara et al., 1985; Hara and Weir, 1986), mouse, hamster, guinea pig and cat (Lee et al., 1978). Other immunohistochemical studies have shown the coexistence of vasoactive intestinal polypeptide (VIP) with AChE within perivascular nerves supplying the cerebral vasculature (Hara et al., 1985; Kobayashi et al., 1983).
By combining histochemical with retrograde tracer techniques and selective denervation, the cerebrovascular parasympathetic innervation has been mapped in the rat, cat, and monkey (Suzuki and Hardebo, 1993). Furthermore, cholinergic nerves were found perivascularly in the adventitia of cerebral vessels, using immunohistochemical techniques with antibodies raised against the ACh synthesizing enzyme choline acetyltransferase (ChAT) (Suzuki and Hardebo, 1993). A slight reduction of ChAT activity and of ACh content of cerebral vessels was observed after bilateral sphenopalatine ganglionectomy in rats (Hara et al., 1989; Dauphin et al., 1991). Data on age-related changes of perivascular cerebrovascular nerves have shown a reduced expression of vasodilator nerves and an increased expression of vasoconstrictor nerves in aged rats (Mione et al., 1988a), but no microanatomical studies were performed to our knowledge on the influence of aging on the density and pattern of cholinergic cerebrovascular nerves. The aim of this study was to investigate age-related changes of cholinergic nerves in the rat pial and intracerebral arteries by immunohistochemical techniques associated with quantitative evaluation. Cholinergic nerves were identified using antibodies raised against ACh. A preliminary account of this work was presented to the 10th Congress of the Italian Group for the Study of Neuromorphology (G.I.S.N.) (El-Assouad and Tayebati, 2001).
2. Materials and methods
2.1. Animals and tissue treatment Male Fisher 344 rats (Charles River, Calco, Italy) aged 6 months (young, n= 10), 15 months (adult, n =10) and 22 months (senescent, n= 10) were used. They were weighed, anaesthetized with ketamine/promazinic acid (4:1) and perfused through the ascending aorta using a peristaltic pump. Infusion solutions were 0.9% NaCl (250 ml) followed by a 0.1% formalin solution (500 ml) freshly prepared from paraformaldehyde powder. At the end of perfusion rats were decapitated, the
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skull was opened and the brain, including pia mater, was rapidly removed. In five rats per age group circle of Willis arteries together with the pia-arachnoid membrane was removed with the aid of a stereomicroscope. Basilar and middle cerebral arteries were dissected out and opened longitudinally, whereas pial vessels of frontal and occipital lobes were left unopened. Vessels were then stretched flat on microscope slides as whole months. In the remaining rats, brains were washed in ice-cold 0.9% NaCl solution and cut in coronal sections of cerebral hemispheres corresponding to head of neostriatum (sections A200– A340 mm) and occipital cortex (sections A480–620 mm) (Ko¨ nig and Klippel, 1963). Slices were included in a cryoprotectant medium, frozen in liquid nitrogen and stored at − 80 °C until used. Sections (16 mm thick) of different brain slices were cut serially at a − 20 °C microtome cryostat and mounted on polylysinated microscope slides.
2.2. Acetylcholine immunohistochemistry Slides were equilibrated at room temperature for 30 –40 min, and washed (3× 15 min) under continuous stirring in a solution A (Tris 0.05 M and 8.5 g/l sodium metabisulphite, pH 7.5). Slides were then transferred in the above solution A to which 3% non-specific calf serum was added and further incubated for 90 min under continuous stirring. Sections were then exposed overnight at 4 °C to a rabbit polyclonal anti-ACh antibody diluted 1:200 in a solution A containing 0.2% Triton X-100 and 1% calf serum (200 ml per section). This antibody concentration was established in a series of preliminary experiments. The slides were then washed (3×10 min) under stirring in a solution B (Tris 0.05 M and 8.5 g/l NaCl, pH 7.5) and subsequently exposed for 60 min at 37 °C to a fluorescein isothiocyanate goat-anti rabbit secondary antibody (for whole mounts only) or to a peroxidase/anti-peroxidase conjugated secondary antibody (for both whole mounts and brain sections. In this second case, a 0.05% 3,3%-diaminobenzidine tetrahydrochloride solution was used as a chromogen for the peroxidase reaction.
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The specificity of immune reaction was assessed by omitting the primary antibody or using a primary antibody pre-adsorbed with ACh. Immunohistochemistry reactions were stopped by putting sections in an ice-cold Tris 0.05 M. The slides were then dehydrated in increasing concentrations of alcohol, cleared in xilene and mounted in Entellan®.
2.3. Quantitati6e analysis The density of cholinergic innervation of whole mounts of basilar and middle cerebral arteries and of pial arteries of frontal and occipital lobes of rats of different age groups was assessed by quantitative image analysis using a IAS 2000 image analyser (Delta Sistemi, Rome, Italy), connected via a TV camera to the microscope. Whole mounts were viewed under a FITC filter-equipped fluorescence microscope using a × 20 objective and × 10 ocular lens. For each vessel of different animals, five fields were randomly selected. In these fields the mean number of nerve fibres× mm − 1 intersecting grid lines along the circumference of the vessel wall and the number of varicosities in a 200 mm length of nerve fibres were calculated (Mione et al., 1988b). Quantitative analysis of the density of innervation of sections of pial and intracerebral arteries was assessed by examining five sections per rat and by identifying in these sections three medium-sized (external diameter 150–50 mm) pial or intracerebral arteries (Amenta, 1991). On these arteries, the density of perivascular nerve bundles was evaluated by measuring directly under the microscope, using a × 20 objective and a × 10 ocular lens the number of axons around the circumference of the vessel wall and the number of nerve varicosities (identified by their spot-like immunoreactivity compared with non-varicose portions of nerve fibres) around the circumference of the vessel wall.
2.4. Chemicals A rabbit ACh-polyclonal antibody (Cat. No. 0030-2004, batch 21010252) was purchased from Biotrend Chemikalien GmbH (Cologne, Ger-
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many). The specificity of this antibody was assessed by ELISA or by immunohistochemistry in sections including septum or nucleus basalis magnocellularis (NBM). Anti-rabbit peroxidase conjugated secondary antibody and other chemicals were products of Sigma Chemical Co. (St. Louis, MO, USA).
2.5. Statistics Means of data of image analysis and of quantitative evaluation of the number of nerve fibres and varicosities around basilar and middle cerebral arteries, pial and intracerebral arteries were calculated per single animal. Group means were derived from single animal data. Values were then analysed statistically by analysis of variance (ANOVA) followed by Duncan’s multiple range test as a post hoc test.
3. Results Whole mounts of circle of basilar and middle cerebral arteries and pial arteries developed fluorescent (Fig. 1A and B) or dark-brown nerve fibre-like structures carrying varicosities. Sections of pial or intracerebral arteries developed perivascular nerve fibre-like structures located in the adventitia and adventitia–media transitional zone (Fig. 1C and D). Data of the density of ACh-immunoreactive nerve fibres in whole mounts of rat basilar and middle cerebral arteries are summarized in Table 1. As shown, in young rats basilar artery and pial arteries of occipital lobe were supplied with a sparser innervation than middle cerebral artery and pial arteries of frontal lobe. The density of ACh-immunoreactive nerve fibres was reduced in adult rats compared with young animals and no
Fig. 1. A and B: Whole mounts of basilar artery obtained from a 6-month-old (young, panel A) and from a 22-month-old (senescent, panel B) rat. ACh immunofluorescence. Note the development of a network-like plexus of fluorescent axons rich in varicosities. Both the density of the plexus and the number of varicosities are decreased in senescent compared to young rats. Calibration bar: 43 mm. C and D: Sections of intracerebral arteries of frontal lobe obtained from a 6-month-old (young, panel A) and from a 22-month-old (senescent, panel B) rat. ACh immunohistochemistry. Note the presence of immunoreactive axons (arrowheads) located perivascularly. The number of these axons is lower in senescent compared to young rats. Calibration bar: 35 mm.
D. El-Assouad, S.K. Tayebati / Mechanisms of Ageing and De6elopment 123 (2002) 529–536 Table 1 ACh-immunoreactive nerve fibres density in whole mounts of basilar and middle cerebral arteries and of frontal or occipital pial arteries Young (n = 5) Basilar artery Nerve fibres 12.7 9 0.9 Number of 30.3 9 2.1 varicosities
pial and intracerebral arteries of adult and senescent rats compared to young animals (Table 2 and Fig. 1D).
Adult (n =5) Senescent (n=5)
4. Discussion 9.05 9 0.4* 23.1 9 1.2*
7.3 90.2** 18.5 91.5**
Middle cerebral artery Nerve fibres 22.6 9 1.2*** 15.3 9 0.8*,*** 16.190.5*,*** Number of 48.4 9 3.5*** 35.1 9 2.4*,*** 38.591.9*,*** varicosities Pial arteries (occipital lobe) Nerve fibres 9.6 9 0.5 Number of 28.1 9 1.8 varicosities
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6.3 9 0.2* 19.5 9 1.9*
7.1 9 0.3* 18.6 9 1.3*
Pial arteries ( frontal lobe) Nerve fibres 18.4 90.9*** 13.2 9 0.8*,*** 12.4 9 0.6*,*** Number of 36.8 92.4*** 27.1 9 1.6*,*** 28.4 9 2.1*,*** varicosities Data are the means 9 SE and were calculated as detailed in Section 2.3. Nerve fibre values are expressed as number of nerve fibres×mm−1 intersecting grid lines along the circumference of the vessel wall. Number of varicosities indicates their number in a 200 mm length of nerve fibres. * PB0.01 vs. young. ** PB0.05 vs. adult. *** PB0.05 vs. basilar artery or pial arteries of occipital lobe.
further reduced in senescent rats with the only exception of basilar artery (Fig. 1B) in which the number of nerve fibres and of varicosities were decreased in senescent compared to adult rats (Table 1). Values of the number of axons and of nerve varicosities around the circumference of the vessel wall in pial and intracerebral arteries are summarized in Table 2. Similarly as found in whole mounts of cerebral or of pial arteries, the density of innervation assessed in sections of anterior pial and intracerebral vessels was greater than that of corresponding arteries located in the occipital lobe (Table 2). The innervation of intracerebral arteries was sparser than that of pial arteries (Table 2). Both the number of ACh-immunoreactive axons and of varicosities were decreased in
As mentioned in the introduction, the presence of cholinergic nerves in the brain vasculature has been a matter of controversy, partly due to the lack of reliable histochemical markers. Recent histochemical and biochemical studies have clearly demonstrated the presence of a cholinergic cerebrovascular innervation and analysed its source and pathways. These studies have shown nerve fibres containing ChAT and VIP in cerebral vessels as well as the coexistence of VIP and AChE in perivascular nerves supplying cerebral vasculature (Suzuki and Hardebo, 1993). ACh Table 2 Number of axons and of nerve varicosities around the circumference of the vessel wall in pial and intracerebral arteries Young (n =5)
Adult (n =5)
Senescent (n =5)
Pial arteries ( frontal lobe) Number of axons 25 9 1.8 Number of 48 92.7 varicosities
17 90.8* 38 91.7*
15 91.0* 35 9 1.9*
Pial arteries (occipital lobe) Number of axons 20 9 1.4** Number of 39 9 2.3** varicosities
14 9 0.6*,** 28 9 1.3*,**
12 9 0.7*,** 25 91.6*,**
Intracerebral arteries ( frontal lobe) Number of axons 15 90.6 11 90.4* Number of 28 91.6 22 90.5* varicosities
10 9 0.6* 23 90.7*
Intracerebral arteries (occipital lobe) Number of axons 12 9 0.6** 9 90.4*,** Number of 20 9 1.5** 15 90.6*,** varicosities
8 90.5*,** 16 9 0.7*,**
Data are the means 9SE and were calculated as detailed in Section 2.3. Number of axons indicates their number around the circumference of the vessel wall. Number of varicosities indicates their number around the circumference of the vessel wall. * PB0.01 vs. young. ** PB0.05 vs. pial or intracerebral arteries of frontal lobe.
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was also assayed in cerebral vessels using biochemical techniques, but no direct demonstration of cerebrovascular ACh-containing nerve fibres is so far available (Suzuki and Hardebo, 1993). In this respect the present study represents the first direct demonstration of ACh immunoreactive axons supplying circle of Willis and pial arteries, as well as intracerebral arteries. ACh is considered an important regulator of local brain CBF (Suzuki and Hardebo, 1993). Cholinergic fibres originating in the NBM of Meynert and in the septal complex project to cerebral cortex and hippocampus, respectively. Their electrical stimulation increases regional CBF in the parietal cortex (Adachi et al., 1992). This effect on CBF is independent by systemic arterial blood pressure (Biesold et al., 1989) and is associated with an increased release of extracellular ACh in both cerebral cortex and hippocampus (Kurosawa et al., 1989a; Sato and Sato, 1995). Hence, stimulation of cholinergic fibres originating in forebrain cholinergic nuclei causes neurogenic vasodilatation in cerebral cortex and hippocampus. The flow increase resulting from direct stimulation of cholinergic fibres is not attenuated by anticholinergic agents, suggesting that it is consequent to a predominant release of VIP or of some other vasodilator agent stored in parasympathetic nerves instead of ACh (Suzuki et al., 1990). Recent studies on cerebrovascular parasympathetic nerves have revealed their source and pathways of distribution (Suzuki and Hardebo, 1993). Apparently cholinergic fibres originating in the NBM vasodilate blood vessels in the cortical parenchymal tissue but not pial arteries (Adachi et al., 1992). Cholinergic supply of pial vessels (Edvinsson, 1975a) originate from sphenopalatine, otic and internal carotid ganglia (Suzuki et al., 1990). The otic ganglion innervates cerebral vessels with fibres running via the superficial and deep petrosal nerves, ascending the internal carotid artery, and then distributing to basal vessels (Shimizu, 1994). Nerve terminals are usually situated in the adventitia, immediately adjacent to the muscular layer of the media. These terminals do not penetrate more than one-third of the media, except in certain areas where terminals have
been found all through the muscular layer (Nielsen et al., 1970). The demonstration of AChimmunoreactive nerve fibres both in circle of Willis and pial arteries and in intracerebral arteries and the observation of a different density of these fibres between extraparenchymal and intracerebral arteries support the above functional data. Cerebrovascular disorders occur more often in the elderly and vascular remodelling and changes in vascular responsiveness are documented in the central nervous system in old age (Paulson and Strandgaard, 1990). A reduction in cerebral arteriolar numerical density, cross-sectional area, distensibility, and relative proportions of cellular elements of cerebral arteriolar wall, as well as a decreased endothelium-dependent relaxation were reported (Salter et al., 1998). A number of correlations were demonstrated between cerebrovascular disease and specific alterations of neurotransmitter systems associated with cerebral vasculature (Dauphin and MacKenzie, 1995). Morphological studies have analysed sympathetic and neuropeptidergic innervation of cerebrovascular tree as a function of age. A decreased expression of perivascular vasoconstrictor nerve fibres consistent with a loss of sympathetic trophic regulation of vascular smooth muscle and an increased susceptibility to sclerotic and degenerative lesions was observed in old age (Mione et al., 1988a). A concomitant increase in VIP- and calcitonin gene related peptide (CGRP)-containing vasodilatory nerve fibres interpreted as a compensatory mechanism for providing a greater blood flow to the brain in old age was also reported in the rat (Mione et al., 1988a). Data on the influence of aging on cholinergic cerebrovascular innervation are limited primarily to functional and biochemical investigations. ChAT activity in the ventral globus pallidus does not differ significantly between young and aged rats (Luine et al., 1986). On the other hand, healthy aged rats maintain the ability to release ACh from the terminals of neurons originating in the NBM, as well as the ability of vasodilating parietal cortical blood vessel after stimulation of the NBM similarly as found in adult rats (Kurosawa et al., 1989b).
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Cholinergic innervation of pial arteries in senescent rats: an immunohistochemical study Dahuk El-Assouad *, Seyed Khosrow Tayebati Sezione di Anatomia Umana, Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Uni6ersita` di Camerino, Via Scalzino, 3, 62032 Camerino, Italy Received 16 February 2001; received in revised form 11 June 2001; accepted 27 July 2001
Abstract Perivascular acetylcholine (ACh)-immunoreactive nerve fibres were demonstrated in basilar and middle cerebral arteries, in pial arteries and arterioles and in intracerebral arteries of male Fisher 344 rats of 6 months (young), 15 months (adult) and 22 months (senescent). Analysis included whole mounts of basilar and middle cerebral arteries, of pial arteries and sections of brain including pia-arachnoid membrane to demonstrate the localization of nerve fibres throughout the wall of pial and of intracerebral arteries. ACh-immunoreactive nerve fibres were demonstrated by indirect immunohistochemistry using a polyclonal anti-ACh antibody and their relative density was quantified. Perivascular ACh-immunoreactive nerve fibres were located in basilar and middle cerebral arteries, in pial arteries and arterioles and in intracerebral arteries. These fibres were found in the adventitia and adventitia– media border with a higher density in pial rather than in intracerebral arteries. A decrease of ACh-immunoreactive nerve fibres was observed both in pial and intracerebral arteries of adult or senescent rats compared to younger cohorts. The direct demonstration of ACh-immunoreactive nerve fibres in the cerebrovascular tree may contribute to evaluate the influence of experimental and pathological conditions on cerebrovascular cholinergic neuroeffector mechanisms, including a role of cholinergic innervation in the pathophysiology of cerebrovascular disease of the elderly. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral arteries; Acetylcholine; Immunohistochemistry; Aging; Fisher 344 rats
1. Introduction The existence of an autonomic nervous control of cerebral vessels was extensively documented by
The present work was awarded the young neuromorphologist G.I.S.N. prize for 2000 to Dr. D. El-Assouad. * Corresponding author. Tel.: +39-0737-403311; fax: + 390737-630618. E-mail address: [email protected] (D. El-Assouad).
histochemical, ultrastructural, and pharmacological studies. The best known autonomic supply of cerebrovascular tree is that provided by sympathetic nervous system. Large and small cerebral arteries, pial and parenchymal arteries, and veins receive a dense sympathetic innervation (Nielsen and Owman, 1967; Purdy and Bevan, 1977), the density of which in different brain areas is rather heterogeneous (Edvinsson, 1975b). Sympathetic neuroeffector junctions release noradrenaline.
0047-6374/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 3 5 2 - 9
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This neurotransmitter has a primarily vasoconstrictor function in the cerebrovascular tree (Kuschinsky and Wahl, 1975; Wei et al., 1975). Sympathetic nerves to cerebral vessels can contain also neuropeptide Y acting as a co-transmitter with noradrenaline. The neurogenic vasoconstriction induced by activity of this subpopulation of nerve fibres is mainly due to the release of neuropeptide Y, whereas noradrenaline or other vasoconstrictor agents do not contribute significantly to the increase of cerebrovascular tone. Cerebrovascular sympathetic nerves containing neuropeptide Y are probably active in cerebral auto-regulation (Branston, 1995), although sympathetic innervation of cerebral vessels influences cerebral blood flow (CBF) mainly under conditions of cerebrovascular stress rather than in normal conditions (Edvinsson et al., 1993; Branston, 1995). The presence of non-sympathetic cholinergic nerve fibres in the brain vasculature has been a matter of controversy. In fact, microanatomical demonstration of parasympathetic nerves in the cerebral blood vessels has been hindered by the lack of suitable markers for the identification of cholinergic nerves. The first studies on the topic used the acetylcholine (ACh) catabolic enzyme acetylcholinesterase (AChE) as a marker of cholinergic nerves. Unfortunately, also some populations of sympathetic nerves express AChE and the enzyme degrades not only ACh, but also some neuropeptides present in autonomic nerves (Amenta et al., 1980b). Hence, AChE cannot be considered as a reliable marker of cholinergic non-sympathetic nerves (Amenta et al., 1980b). Perivascular AChE-positive nerve fibres were visualized in cerebral blood vessels of several species including humans (Edvinsson et al., 1976), monkey (Denn and Stone, 1976), dog (Amenta et al., 1980a), rabbit (Edvinsson et al., 1972; Baramidze et al., 1982), rat (Edvinsson et al., 1972; Licata et al., 1975; Vasquez and Purves, 1979; Kobayashi et al., 1983; Hara et al., 1985; Hara and Weir, 1986), mouse, hamster, guinea pig and cat (Lee et al., 1978). Other immunohistochemical studies have shown the coexistence of vasoactive intestinal polypeptide (VIP) with AChE within perivascular nerves supplying the cerebral vasculature (Hara et al., 1985; Kobayashi et al., 1983).
By combining histochemical with retrograde tracer techniques and selective denervation, the cerebrovascular parasympathetic innervation has been mapped in the rat, cat, and monkey (Suzuki and Hardebo, 1993). Furthermore, cholinergic nerves were found perivascularly in the adventitia of cerebral vessels, using immunohistochemical techniques with antibodies raised against the ACh synthesizing enzyme choline acetyltransferase (ChAT) (Suzuki and Hardebo, 1993). A slight reduction of ChAT activity and of ACh content of cerebral vessels was observed after bilateral sphenopalatine ganglionectomy in rats (Hara et al., 1989; Dauphin et al., 1991). Data on age-related changes of perivascular cerebrovascular nerves have shown a reduced expression of vasodilator nerves and an increased expression of vasoconstrictor nerves in aged rats (Mione et al., 1988a), but no microanatomical studies were performed to our knowledge on the influence of aging on the density and pattern of cholinergic cerebrovascular nerves. The aim of this study was to investigate age-related changes of cholinergic nerves in the rat pial and intracerebral arteries by immunohistochemical techniques associated with quantitative evaluation. Cholinergic nerves were identified using antibodies raised against ACh. A preliminary account of this work was presented to the 10th Congress of the Italian Group for the Study of Neuromorphology (G.I.S.N.) (El-Assouad and Tayebati, 2001).
2. Materials and methods
2.1. Animals and tissue treatment Male Fisher 344 rats (Charles River, Calco, Italy) aged 6 months (young, n= 10), 15 months (adult, n =10) and 22 months (senescent, n= 10) were used. They were weighed, anaesthetized with ketamine/promazinic acid (4:1) and perfused through the ascending aorta using a peristaltic pump. Infusion solutions were 0.9% NaCl (250 ml) followed by a 0.1% formalin solution (500 ml) freshly prepared from paraformaldehyde powder. At the end of perfusion rats were decapitated, the
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skull was opened and the brain, including pia mater, was rapidly removed. In five rats per age group circle of Willis arteries together with the pia-arachnoid membrane was removed with the aid of a stereomicroscope. Basilar and middle cerebral arteries were dissected out and opened longitudinally, whereas pial vessels of frontal and occipital lobes were left unopened. Vessels were then stretched flat on microscope slides as whole months. In the remaining rats, brains were washed in ice-cold 0.9% NaCl solution and cut in coronal sections of cerebral hemispheres corresponding to head of neostriatum (sections A200– A340 mm) and occipital cortex (sections A480–620 mm) (Ko¨ nig and Klippel, 1963). Slices were included in a cryoprotectant medium, frozen in liquid nitrogen and stored at − 80 °C until used. Sections (16 mm thick) of different brain slices were cut serially at a − 20 °C microtome cryostat and mounted on polylysinated microscope slides.
2.2. Acetylcholine immunohistochemistry Slides were equilibrated at room temperature for 30 –40 min, and washed (3× 15 min) under continuous stirring in a solution A (Tris 0.05 M and 8.5 g/l sodium metabisulphite, pH 7.5). Slides were then transferred in the above solution A to which 3% non-specific calf serum was added and further incubated for 90 min under continuous stirring. Sections were then exposed overnight at 4 °C to a rabbit polyclonal anti-ACh antibody diluted 1:200 in a solution A containing 0.2% Triton X-100 and 1% calf serum (200 ml per section). This antibody concentration was established in a series of preliminary experiments. The slides were then washed (3×10 min) under stirring in a solution B (Tris 0.05 M and 8.5 g/l NaCl, pH 7.5) and subsequently exposed for 60 min at 37 °C to a fluorescein isothiocyanate goat-anti rabbit secondary antibody (for whole mounts only) or to a peroxidase/anti-peroxidase conjugated secondary antibody (for both whole mounts and brain sections. In this second case, a 0.05% 3,3%-diaminobenzidine tetrahydrochloride solution was used as a chromogen for the peroxidase reaction.
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The specificity of immune reaction was assessed by omitting the primary antibody or using a primary antibody pre-adsorbed with ACh. Immunohistochemistry reactions were stopped by putting sections in an ice-cold Tris 0.05 M. The slides were then dehydrated in increasing concentrations of alcohol, cleared in xilene and mounted in Entellan®.
2.3. Quantitati6e analysis The density of cholinergic innervation of whole mounts of basilar and middle cerebral arteries and of pial arteries of frontal and occipital lobes of rats of different age groups was assessed by quantitative image analysis using a IAS 2000 image analyser (Delta Sistemi, Rome, Italy), connected via a TV camera to the microscope. Whole mounts were viewed under a FITC filter-equipped fluorescence microscope using a × 20 objective and × 10 ocular lens. For each vessel of different animals, five fields were randomly selected. In these fields the mean number of nerve fibres× mm − 1 intersecting grid lines along the circumference of the vessel wall and the number of varicosities in a 200 mm length of nerve fibres were calculated (Mione et al., 1988b). Quantitative analysis of the density of innervation of sections of pial and intracerebral arteries was assessed by examining five sections per rat and by identifying in these sections three medium-sized (external diameter 150–50 mm) pial or intracerebral arteries (Amenta, 1991). On these arteries, the density of perivascular nerve bundles was evaluated by measuring directly under the microscope, using a × 20 objective and a × 10 ocular lens the number of axons around the circumference of the vessel wall and the number of nerve varicosities (identified by their spot-like immunoreactivity compared with non-varicose portions of nerve fibres) around the circumference of the vessel wall.
2.4. Chemicals A rabbit ACh-polyclonal antibody (Cat. No. 0030-2004, batch 21010252) was purchased from Biotrend Chemikalien GmbH (Cologne, Ger-
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many). The specificity of this antibody was assessed by ELISA or by immunohistochemistry in sections including septum or nucleus basalis magnocellularis (NBM). Anti-rabbit peroxidase conjugated secondary antibody and other chemicals were products of Sigma Chemical Co. (St. Louis, MO, USA).
2.5. Statistics Means of data of image analysis and of quantitative evaluation of the number of nerve fibres and varicosities around basilar and middle cerebral arteries, pial and intracerebral arteries were calculated per single animal. Group means were derived from single animal data. Values were then analysed statistically by analysis of variance (ANOVA) followed by Duncan’s multiple range test as a post hoc test.
3. Results Whole mounts of circle of basilar and middle cerebral arteries and pial arteries developed fluorescent (Fig. 1A and B) or dark-brown nerve fibre-like structures carrying varicosities. Sections of pial or intracerebral arteries developed perivascular nerve fibre-like structures located in the adventitia and adventitia–media transitional zone (Fig. 1C and D). Data of the density of ACh-immunoreactive nerve fibres in whole mounts of rat basilar and middle cerebral arteries are summarized in Table 1. As shown, in young rats basilar artery and pial arteries of occipital lobe were supplied with a sparser innervation than middle cerebral artery and pial arteries of frontal lobe. The density of ACh-immunoreactive nerve fibres was reduced in adult rats compared with young animals and no
Fig. 1. A and B: Whole mounts of basilar artery obtained from a 6-month-old (young, panel A) and from a 22-month-old (senescent, panel B) rat. ACh immunofluorescence. Note the development of a network-like plexus of fluorescent axons rich in varicosities. Both the density of the plexus and the number of varicosities are decreased in senescent compared to young rats. Calibration bar: 43 mm. C and D: Sections of intracerebral arteries of frontal lobe obtained from a 6-month-old (young, panel A) and from a 22-month-old (senescent, panel B) rat. ACh immunohistochemistry. Note the presence of immunoreactive axons (arrowheads) located perivascularly. The number of these axons is lower in senescent compared to young rats. Calibration bar: 35 mm.
D. El-Assouad, S.K. Tayebati / Mechanisms of Ageing and De6elopment 123 (2002) 529–536 Table 1 ACh-immunoreactive nerve fibres density in whole mounts of basilar and middle cerebral arteries and of frontal or occipital pial arteries Young (n = 5) Basilar artery Nerve fibres 12.7 9 0.9 Number of 30.3 9 2.1 varicosities
pial and intracerebral arteries of adult and senescent rats compared to young animals (Table 2 and Fig. 1D).
Adult (n =5) Senescent (n=5)
4. Discussion 9.05 9 0.4* 23.1 9 1.2*
7.3 90.2** 18.5 91.5**
Middle cerebral artery Nerve fibres 22.6 9 1.2*** 15.3 9 0.8*,*** 16.190.5*,*** Number of 48.4 9 3.5*** 35.1 9 2.4*,*** 38.591.9*,*** varicosities Pial arteries (occipital lobe) Nerve fibres 9.6 9 0.5 Number of 28.1 9 1.8 varicosities
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6.3 9 0.2* 19.5 9 1.9*
7.1 9 0.3* 18.6 9 1.3*
Pial arteries ( frontal lobe) Nerve fibres 18.4 90.9*** 13.2 9 0.8*,*** 12.4 9 0.6*,*** Number of 36.8 92.4*** 27.1 9 1.6*,*** 28.4 9 2.1*,*** varicosities Data are the means 9 SE and were calculated as detailed in Section 2.3. Nerve fibre values are expressed as number of nerve fibres×mm−1 intersecting grid lines along the circumference of the vessel wall. Number of varicosities indicates their number in a 200 mm length of nerve fibres. * PB0.01 vs. young. ** PB0.05 vs. adult. *** PB0.05 vs. basilar artery or pial arteries of occipital lobe.
further reduced in senescent rats with the only exception of basilar artery (Fig. 1B) in which the number of nerve fibres and of varicosities were decreased in senescent compared to adult rats (Table 1). Values of the number of axons and of nerve varicosities around the circumference of the vessel wall in pial and intracerebral arteries are summarized in Table 2. Similarly as found in whole mounts of cerebral or of pial arteries, the density of innervation assessed in sections of anterior pial and intracerebral vessels was greater than that of corresponding arteries located in the occipital lobe (Table 2). The innervation of intracerebral arteries was sparser than that of pial arteries (Table 2). Both the number of ACh-immunoreactive axons and of varicosities were decreased in
As mentioned in the introduction, the presence of cholinergic nerves in the brain vasculature has been a matter of controversy, partly due to the lack of reliable histochemical markers. Recent histochemical and biochemical studies have clearly demonstrated the presence of a cholinergic cerebrovascular innervation and analysed its source and pathways. These studies have shown nerve fibres containing ChAT and VIP in cerebral vessels as well as the coexistence of VIP and AChE in perivascular nerves supplying cerebral vasculature (Suzuki and Hardebo, 1993). ACh Table 2 Number of axons and of nerve varicosities around the circumference of the vessel wall in pial and intracerebral arteries Young (n =5)
Adult (n =5)
Senescent (n =5)
Pial arteries ( frontal lobe) Number of axons 25 9 1.8 Number of 48 92.7 varicosities
17 90.8* 38 91.7*
15 91.0* 35 9 1.9*
Pial arteries (occipital lobe) Number of axons 20 9 1.4** Number of 39 9 2.3** varicosities
14 9 0.6*,** 28 9 1.3*,**
12 9 0.7*,** 25 91.6*,**
Intracerebral arteries ( frontal lobe) Number of axons 15 90.6 11 90.4* Number of 28 91.6 22 90.5* varicosities
10 9 0.6* 23 90.7*
Intracerebral arteries (occipital lobe) Number of axons 12 9 0.6** 9 90.4*,** Number of 20 9 1.5** 15 90.6*,** varicosities
8 90.5*,** 16 9 0.7*,**
Data are the means 9SE and were calculated as detailed in Section 2.3. Number of axons indicates their number around the circumference of the vessel wall. Number of varicosities indicates their number around the circumference of the vessel wall. * PB0.01 vs. young. ** PB0.05 vs. pial or intracerebral arteries of frontal lobe.
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was also assayed in cerebral vessels using biochemical techniques, but no direct demonstration of cerebrovascular ACh-containing nerve fibres is so far available (Suzuki and Hardebo, 1993). In this respect the present study represents the first direct demonstration of ACh immunoreactive axons supplying circle of Willis and pial arteries, as well as intracerebral arteries. ACh is considered an important regulator of local brain CBF (Suzuki and Hardebo, 1993). Cholinergic fibres originating in the NBM of Meynert and in the septal complex project to cerebral cortex and hippocampus, respectively. Their electrical stimulation increases regional CBF in the parietal cortex (Adachi et al., 1992). This effect on CBF is independent by systemic arterial blood pressure (Biesold et al., 1989) and is associated with an increased release of extracellular ACh in both cerebral cortex and hippocampus (Kurosawa et al., 1989a; Sato and Sato, 1995). Hence, stimulation of cholinergic fibres originating in forebrain cholinergic nuclei causes neurogenic vasodilatation in cerebral cortex and hippocampus. The flow increase resulting from direct stimulation of cholinergic fibres is not attenuated by anticholinergic agents, suggesting that it is consequent to a predominant release of VIP or of some other vasodilator agent stored in parasympathetic nerves instead of ACh (Suzuki et al., 1990). Recent studies on cerebrovascular parasympathetic nerves have revealed their source and pathways of distribution (Suzuki and Hardebo, 1993). Apparently cholinergic fibres originating in the NBM vasodilate blood vessels in the cortical parenchymal tissue but not pial arteries (Adachi et al., 1992). Cholinergic supply of pial vessels (Edvinsson, 1975a) originate from sphenopalatine, otic and internal carotid ganglia (Suzuki et al., 1990). The otic ganglion innervates cerebral vessels with fibres running via the superficial and deep petrosal nerves, ascending the internal carotid artery, and then distributing to basal vessels (Shimizu, 1994). Nerve terminals are usually situated in the adventitia, immediately adjacent to the muscular layer of the media. These terminals do not penetrate more than one-third of the media, except in certain areas where terminals have
been found all through the muscular layer (Nielsen et al., 1970). The demonstration of AChimmunoreactive nerve fibres both in circle of Willis and pial arteries and in intracerebral arteries and the observation of a different density of these fibres between extraparenchymal and intracerebral arteries support the above functional data. Cerebrovascular disorders occur more often in the elderly and vascular remodelling and changes in vascular responsiveness are documented in the central nervous system in old age (Paulson and Strandgaard, 1990). A reduction in cerebral arteriolar numerical density, cross-sectional area, distensibility, and relative proportions of cellular elements of cerebral arteriolar wall, as well as a decreased endothelium-dependent relaxation were reported (Salter et al., 1998). A number of correlations were demonstrated between cerebrovascular disease and specific alterations of neurotransmitter systems associated with cerebral vasculature (Dauphin and MacKenzie, 1995). Morphological studies have analysed sympathetic and neuropeptidergic innervation of cerebrovascular tree as a function of age. A decreased expression of perivascular vasoconstrictor nerve fibres consistent with a loss of sympathetic trophic regulation of vascular smooth muscle and an increased susceptibility to sclerotic and degenerative lesions was observed in old age (Mione et al., 1988a). A concomitant increase in VIP- and calcitonin gene related peptide (CGRP)-containing vasodilatory nerve fibres interpreted as a compensatory mechanism for providing a greater blood flow to the brain in old age was also reported in the rat (Mione et al., 1988a). Data on the influence of aging on cholinergic cerebrovascular innervation are limited primarily to functional and biochemical investigations. ChAT activity in the ventral globus pallidus does not differ significantly between young and aged rats (Luine et al., 1986). On the other hand, healthy aged rats maintain the ability to release ACh from the terminals of neurons originating in the NBM, as well as the ability of vasodilating parietal cortical blood vessel after stimulation of the NBM similarly as found in adult rats (Kurosawa et al., 1989b).
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