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Cerebral small vessel disease predisposes to temporal lobe epilepsy in spontaneously hypertensive rats
Accepted Manuscript Title: Cerebral small vessel disease predisposes to temporal lobe epilepsy in spontaneously hypertensive rats Authors: Emilio Russo, Antonio Leo, Francesca Scicchitano, Annalidia Donato, Edoardo Ferlazzo, Sara Gasparini, Vittoria Cianci, Chiara Mignogna, Giuseppe Donato, Rita Citraro, Umberto Aguglia, Giovambattista De Sarro PII: DOI: Reference:
S0361-9230(17)30001-1 http://dx.doi.org/doi:10.1016/j.brainresbull.2017.02.003 BRB 9170
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
1-1-2017 30-1-2017 13-2-2017
Please cite this article as: Emilio Russo, Antonio Leo, Francesca Scicchitano, Annalidia Donato, Edoardo Ferlazzo, Sara Gasparini, Vittoria Cianci, Chiara Mignogna, Giuseppe Donato, Rita Citraro, Umberto Aguglia, Giovambattista De Sarro, Cerebral small vessel disease predisposes to temporal lobe epilepsy in spontaneously hypertensive rats, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2017.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cerebral small vessel disease predisposes to temporal lobe epilepsy in spontaneously hypertensive rats
Running Title: CSVD and Epilepsy
Emilio Russo1,*, Antonio Leo1, Francesca Scicchitano1, Annalidia Donato1, Edoardo Ferlazzo2,3, Sara Gasparini2,3, Vittoria Cianci2, Chiara Mignogna1, Giuseppe Donato1, Rita Citraro1, Umberto Aguglia2,3, Giovambattista De Sarro1
1
Science of Health Department, School of Medicine, University “Magna Graecia” of Catanzaro,
Italy; 2
Regional Epilepsy Centre, Bianchi-Melacrino-Morelli Hospital, Reggio Calabria, Italy;
3
Medical and Surgical Sciences Department, School of Medicine, Magna Græcia University of
Catanzaro, Viale Europa, Catanzaro, Italy.
*Author for correspondence: Prof. Emilio Russo Chair of Pharmacology, Department of Science of Health, School of Medicine, University of Catanzaro, Italy Via T. Campanella, 115; 88100 Catanzaro, ITALY. Phone +39 0961 3694191; Fax +39 0961 3694192; e-mail: [email protected]
Highlights
5-weeks-old spontaneously hypertensive rats (SHRs) have similar seizure susceptibility to control rats 16-weeks-old SHRs develop amygdala kindling more rapidly (less stimulations) than control rats 1
16-weeks-old SHRs display cerebral small vessels disease (CSVD) which is prevented by enalapril long-term treatment. Enalapril treatment, inhibiting the development of CSVD, normalizes amygdala kindling development in SHRs CSVD in SHRs is associated with a higher susceptibility to amygdala kindling.
Abstract The link between cerebral small vessel disease (CSVD) and epilepsy has been poorly investigated. Some reports suggest that CSVD may predispose to temporal lobe epilepsy (TLE). Aim of this study was to evaluate whether spontaneously hypertensive rats (SHRs), an established model of systemic hypertension and CSVD, have a propensity to develop TLE more than generalized seizures. To this aim, amygdala kindling, as a model of TLE, and pentylenetetrazole (PTZ)-induced kindling, as a model of generalized seizures, have been used to ascertain whether SHRs are more prone to TLE as compared to Wistar Kyoto control rats. While young SHRs (without CSVD) do not differ from their age-matched controls in both models, old SHRs (with CSVD) develop stage 5 seizures in the amygdala kindling model (TLE) faster than age-matched control rats without CSVD. At odds, no differences between old SHRs and age-matched controls was observed in the development of PTZ kindling. Enalapril pre-treatment prevented the development of CSVD and normalized kindling development to control levels in SHRs. No difference was observed in the response to pharmacological treatment with carbamazepine or losartan. Overall, our study suggests that uncontrolled hypertension leading to CSVD might represent a risk factor for TLE. Further experimental studies are needed to unravel other risk factors that, along with CSVD, may predispose to TLE.
Keywords: SHRs; stroke; hypertension; kindling; pentylenetetrazole.
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1. Introduction Experimental and clinical studies suggest that systemic hypertension and cerebral small vessel disease (CSVD) increase the risk of epilepsy even in the absence of prior clinically detected stroke [5, 17, 23, 24]. CSVD has received little attention until modern brain imaging technologies allowed to detect small deep infarcts and white matter rarefaction (i.e. leukoaraiosis) [25]. Leukoaraiosis may be observed in adults with otherwise unexplained new-onset epilepsy, but it is not clear whether leukoaraiosis represents a mere incidental radiologic finding (i.e. epiphenomenon) or plays an epileptogenic role [8, 9]. Clinical studies suggest that temporal lobe epilepsy (TLE) predominates in epileptic patients with leukoaraiosis [9, 16]. A link between TLE and alterations in the function of renin-angiotensin system (RAS) has been hypothesized [21, 22, 26]. Indeed, type 1 and 2 angiotensin receptors (AT) were found to be up-regulated in the cortex and hippocampus of patients with TLE [1]; moreover, AT-converting enzyme inhibitors (ACEi) displayed antiepileptic activity in some animal models of seizures/epilepsy [7, 19, 21]. Finally, previous reports indicate that spontaneously hypertensive rats (SHRs) could be considered as a suitable tool to study the link between hypertension and epilepsy [37, 38] and, notably, CSVD was observed in this strain [13]. Of note, SHRs display CSVD as a consequence of uncontrolled hypertension [3] and may therefore be used to study the comorbidity between hypertension, CSVD and epilepsy [29]. The primary aim of this study was to evaluate the propensity and possible differences of SHRs to develop either TLE by inducing amygdala kindling (a TLE model) or pentylenetetrazole (PTZ) kindling (a generalized seizures model) in 16-week-old SHRs and age-matched Wistar Kyoto (WKY) control rats. The secondary aims were: 1) to assess the role of ACEi to prevent hypertension-related CSVD and epilepsy by administrating enalapril before both types of kindling in the two strains; 2) to evaluate the role of hypertension or genetic factors in TLE development before CSVD onset by applying the same protocols in 5-week-old SHR and in age-matched WKY
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control rats; 3) to test seizure response to losartan vs carbamazepine in amygdala-kindled rats of both strains.
2. Materials and Methods 2.1 Animals A total of 64 male SHRs and 64 male WKY rats of 3 weeks of age were obtained from Charles River (Milan, Italy). Rats were housed in stable conditions of humidity (60 ± 5%) and temperature (21 ± 2°C), kept under a reversed light/dark (12/12 h) cycle (light on at 19:00 h) and given free access to food and water until the time of experiments. The experimental procedures were carried out in accordance with the guidance and general recommendations with international and national law and policies (EU Directive 2010/63/EU for animal experiments, ARRIVE guidelines and the Basel declaration including the 3R concept) and after approval by the local ethical committee. Measures were included in the protocols to minimize pain and discomfort to the animals and minimize animal usage.
2.2 Drugs Pentylenetetrazole (PTZ; Sigma, Milan, Italy) was dissolved in 0.9% saline and injected s.c. in a volume of 1.0 mL/kg, at a subconvulsive dose of 30 mg/kg every other day until development of kindling or up to 8 weeks [6]. Enalapril (Germed Pharma S.p.A Cinisello Balsamo, Italy) was orally administered at a dose of 10 mg/kg/day by dissolving 20 mg in 120 ml of consumable water (calculated on the basis of the knowledge that rats drink on average 10–12 ml/100 g/day) [4]. The dose of enalapril was based on previous reports and a short preliminary experiment with two doses (10 and 20 mg/kg; n=3 per
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group) administered for 10 days in which the highest dose appeared to be not well-tolerated following a behavioral evaluation by an expert veterinary [2]. Losartan (Sandoz; Varese, Italy), at the dose of 50 mg/kg i.p., was dissolved in 0.9% saline. Carbamazepine (Sigma-Aldrich Milan, Italy), at the dose of 50 mg/kg i.p., was dissolved in dimethyl sulfoxide (DMSO).
2.3 Surgery and electrical kindling procedure Thirty-two SHRs and 32 WKY rats were anesthetized by a tiletamine/zolazepam mixture (1:1; Zoletil 100®; 50 mg/kg i.p.; VIRBAC Srl, Milan, Italy); a bipolar electrode was implanted into the right hemisphere in the basolateral amygdala (AP= 2.2; L= 4.8; H= 8.5 in mm relative to bregma) according to Paxinos and Watson [27], as previously described [18]. All rats after surgery were randomly divided into 8 groups (n=8 for all groups; see Figure 1 for an experimental protocol scheme): 1) WKY of 5 weeks of age (5WKY); 2) WKY of 5 weeks of age treated with enalapril 10 mg/kg/day o.s. (5WKYen); 3) SHR of 5 weeks of age (5SHR); 4) SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s. (5SHRen); 5) WKY of 16 weeks of age (16WKY); 6) WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s. (16WKYen); 7) SHR of 16 weeks of age (16SHR); 8) SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s. (16SHRen). The administration of enalapril was started in all treated groups at 30 days of age and continued up to end of the experimental protocol (e.g. acquisition of kindling status). Electrical stimulation (Grass S88K stimulator) of the amygdala was started after a recovery period of at least 1 week in all groups (range 7-10 days). Afterdischarge Threshold (ADT) was determined, using stimuli of increasing intensity (20μA steps starting from 10μA) presented at 1 min intervals until an AD occurred, the day before the first day of the stimulation period and it was defined as the lowest electrical stimulus that elicited an afterdischarge (AD) lasting at least 3s [18]. From the next
5
day on, pulsed electrical stimulation (500 μA, 1 ms, monophasic square-wave pulses, 50Hz for 1 s) were delivered at intervals of 1 day until 5 consecutive fully kindled seizures (stage 5; seizure severity classified by Racine scale) were elicited [18, 30, 31]. AD duration was also measured on the first day of stimulation and after 5 stage 5 seizures. To test a putative potential difference in pharmacological response between kindled rats of the two different strains, two days after reaching a fully kindled state of 5 stage 5 seizures, the effect on the behavioral seizure score of a single dose of losartan (50 mg/kg) or carbamazepine (50 mg/kg) administered i.p. was assessed. Rats were tested at 60 min after drug administration. The doses of losartan and carbamazepine are consistent with efficacious doses of these drugs in standard kindling and other rodent seizure models [15, 20].
2.4 Pentylenetetrazole Kindling protocol Chemical kindling was induced in 32 SHRs and in 32 WKY rats by pentylenetetrazole (PTZ, 30 mg/kg s.c.) injected every other day at the most up to 8 consecutive weeks in the morning between 9:00 and 11:00 [6]. Animals were randomly divided into 8 groups (n= 8for all groups) as above described for electrical kindling and also in this case 4 out of 8 groups received enalapril (10 mg/kg/day) during the entire time-window of PTZ administration (see Figure 1). After PTZ administration, rats were placed in a Plexiglas box (100 cm wide × 100 cm long × 50 cm high) and observed for 30 min and seizures scored according to the following scale: 0 = no change in behavior; 0.5 = Abnormal behavior (sniffing, extensive washing, orientation); l = isolate myoclonic jerks; 2 = Atypical (unilateral or incomplete) clonic seizures; 3 = fully developed bilateral forelimb clonus; 3.5 = forelimb clonus with a tonic component and twist of body; 4 = Tonic-clonic seizure with suppressed tonic phase; only clonus of all limbs; 5 = fully developed tonic-clonic seizures [6, 40]. The maximum response was recorded for each animal. Rats were
6
considered fully kindled when exhibiting 3 consecutive stage 5 seizures, and then the treatment was discontinued.
2.5 Histological analysis Immediately after either electrical stimulation or PTZ administration, rats were anesthetized with a mixture of tiletamine/zolazepam (1:1; Zoletil 100®; 50 mg/kg, i.p.; VIRBAC Srl, Milan, Italy) and intracardiacally infused with cold phosphate-buffered saline (PBS, pH 7.4) and, consequently, with cold mixture of 4% paraformaldehyde (PFA), 0.2% saturated picric acid, and PBS. Brains were removed and post-fixed overnight at 4° C in the same fixative solution. After paraffin embedding, tissue sections of 4m representative of a coronal cut on bregma were obtained and sections were stained with eosin-hematoxylin for morphology and Luxol fast blue (Klüver Barrera method staining kit; Bioptica; Milan, Italy; Code: W01030799), as previously described [14, 32]. Cortical and subcortical structures were examined for signs of arteriolar hyalinosis and/or vascular damage.
2.6 Statistical analysis Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA 92037, USA). The two age subgroups (5 and 16 weeks old rats) were always analyzed separately and not compared. The ADT and AD duration within age groups was compared with a two-way (factors = strain and treatment) ANOVA for repeated measures followed by Tukey’s post hoc test. Significance of differences in scores of stage 5 seizures were compared using two-way (factors = strain and treatment) ANOVA followed by Bonferroni’s post-hoc test.
3. Results 3.1 Electrical Kindling in SHRs and WKY rats 7
ADT was not significantly different among all groups both on stimulation day 1 and after 5 stage 5 seizures (Figure 2A). AD duration was always significantly longer after kindling development and it was not significantly different between groups with the only exception of 16-week-old untreated SHRs which had a significantly (p<0.05) longer AD on the first day (ADT determination day) in comparison to all other 16-week-old groups (Figure 2B). In addition, 16-week-old untreated SHRs had a significantly faster rate of kindling development (number of AD stimulations: 14 vs 19; p < 0.0001) in comparison to age-matched WKY rats; enalapril normalized kindling development in this strain/group (Figures 3A, B). Interestingly, 5-week-old SHRs did not kindle before agematched WKY rats, and treatment did not influence outcome in all 5-week-old strains (Figure 3B). Carbamazepine reduced seizure score (from an average 5 to 3) in all 16-week-old kindled strains, while losartan had no effects (data not shown).
3.2 PTZ Kindling development Kindled seizures were directly proportional and cumulative to repeated PTZ administration in all strains. None of the animals convulsed on the first administration. No difference in seizure score was found between SHRs and controls at all ages. Both treated and untreated 16-week-old SHR had a similar rate of kindling development in comparison to age-matched WKY rats (Figures 4A, B). 5week-old SHRs did not kindle before age-matched WKY rats, and treatment did not influence kindling process (Figure 4B). In particular, the 3rd stage 5 seizure was achieved after 18 PTZ administrations in all 16-week-old strains and after 16 PTZ administrations in all 5-week-old strains (Figure 4A, B).
3.3 Histological analysis No signs of CSVD were present in all 5-week-old groups (age at analysis: 8 weeks of age in the amygdala kindling groups and 10 weeks of age in the PTZ groups). In the 16-week-old groups, untreated WKY rats did not show CSVD after both PTZ- and amygdala- kindling (age at analysis: 8
20 weeks of age in the amygdala kindling and 22 in the PTZ kindling groups: Figure 5A, D). At odds, untreated SHRs had clear signs of CSVD after both PTZ- and amygdala- kindling (age at analysis: 19 weeks of age in the amygdala kindling and 22 in the PTZ kindling groups: Figure 5B, E). CSVD was uniformly distributed in the cortex, basal ganglia, thalami and subcortical white matter. No interlobar differences in CSVD distribution were observed. Notably, no signs of CSVD were present in enalapril-treated SHRs groups after both PTZ- and amygdala- kindling (age at analysis: 20 weeks of age in the amygdala kindling and 22 in the PTZ kindling groups: Figure 5C, F).
4. Discussion We found that SHRs with hypertension-related CSVD have a higher susceptibility to TLE, as demonstrated by a significantly higher propensity to develop seizures by amygdala kindling compared to WKY control rats. Previous studies suggested that SHRs have a higher susceptibility to tonic extensor seizures [10] and TLE [11, 33, 35, 36], but the exact role of CSVD was not evaluated. We hypothesized that CSVD might play an epileptogenic role. Actually, an early treatment with enalapril prevented the development of CSVD and reduced the propensity of SHRs to develop amygdala-kindled seizures to the same levels revealed in control rats. Altogether, these findings are in keeping with human evidence showing that TLE predominates in epileptic patients with leukoaraiosis [9]. However, our neuropathological data do not clarify the pathogenic role of hypertension-related CSVD on TLE, since small vessel damage did not predominate in temporal lobes of 16-week-old SHRs. Further functional, neuropathological and molecular studies are needed to disentangle this issue. Hypertension has been suggested as an independent risk factor for epilepsy in human studies, although a specific predisposition for TLE has never been demonstrated. Ng et al. [24] found that a history of systemic hypertension represented a risk factor (OR = 1.57) for epilepsy in a case-control 9
study. Hesdorffer et al. [12] found that severe uncontrolled hypertension was associated with an 11fold increased risk of unprovoked seizures in a cross-sectional, case-control study. Finally, Li et al. [17] showed that chronic hypertension (evaluated as left ventricular hypertrophy) represented an independent risk factor (OR = 2.9) for epilepsy in a cross-sectional, case-control study. In our study, we found that only 16-week-old SHRs (with CSVD) had a higher propensity to develop seizures by amygdala kindling as compared to control rats, while 5-week-old SHRs (without CSVD) did not differ from control rats. Interestingly, upregulation of AT1 and AT2 receptors has been found in the hippocampus of patients who underwent temporal lobectomy for refractory epilepsy, suggesting the involvement of renin-angiotensin system in epileptogenesis in these patients [36]. Animal studies corroborate the evidence that an independent renin-angiotensin system exists in the central nervous system and that it may be involved in seizure susceptibility [28, 34, 36, 39]. Tchekalarova et al. [34, 39] showed that the subchronic (14 days) or long-term (16 weeks) treatment with losartan (10 mg/kg) after kainate-induced status epilepticus (SE) reduce seizure frequency and duration, and it may have a neuroprotective effect in the CA3 hippocampus area and septo temporal hilus of the dentate gyrus in SHRs. In our study, losartan was ineffective to reduce seizure severity in amygdala-kindled strains. These findings do not support an antiepileptic effect of losartan in these rat model. Moreover, a single partially effective dose of carbamazepine (50mg/kg) was used to determine whether kindled seizures in SHRs would have a possible different pharmacological responsiveness; no differences were observed, therefore, temporal lobe seizures in the two strains have a likely identical drug sensitivity. Finally, the role played by the genetic predisposition of SHRs for TLE seems to be irrelevant since 5-week-old SHRs without CSVD have not a higher susceptibility to develop amygdala kindled seizures as compared to age-matched control rats.
4.1 Conclusions 10
Overall, our study suggests that uncontrolled hypertension leading to CSVD or small vessels damage might represent a risk factor for the development of TLE more than other epilepsy types. Further studies are needed to clarify whether this result is clinically relevant and to understand which might be other risk factors that need to be combined to CSVD in order to develop TLE as suggested by previous clinical studies [8, 9, 23].
Conflict of interest Authors have nothing to declare.
Acknowledgements None
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[37] J. Tchekalarova, D. Pechlivanova, T. Atanasova, P. Markova, V. Lozanov, A. Stoynev, Diurnal variations in depression-like behavior of Wistar and spontaneously hypertensive rats in the kainate model of temporal lobe epilepsy, Epilepsy Behav, 20 (2011) 277-285. [38] J. Tchekalarova, D. Pechlivanova, D. Itzev, N. Lazarov, P. Markova, A. Stoynev, Diurnal rhythms of spontaneous recurrent seizures and behavioral alterations of Wistar and spontaneously hypertensive rats in the kainate model of epilepsy, Epilepsy Behav, 17 (2010) 23-32. [39] J.D. Tchekalarova, N. Ivanova, D. Atanasova, D.M. Pechlivanova, N. Lazarov, L. Kortenska, R. Mitreva, V. Lozanov, A. Stoynev, Long-Term Treatment with Losartan Attenuates Seizure Activity and Neuronal Damage Without Affecting Behavioral Changes in a Model of Co-morbid Hypertension and Epilepsy, Cellular and molecular neurobiology, 36 (2016) 927-941. [40] J. Veliskova, Behavioral Characterization of Seizures in Rats, in: A. Pitkanen, P.A. Schwartzkroin, S.L. Moshe (Eds.) Models of Seizures and Epilepsy, Elsevier Inc., 2006, pp. 601611.
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Figure Legends
Figure 1. Scheme of the experimental protocol used in the study. SHRs and WKY rats of the two ages considered (5 and 16 weeks) were divided in two subgroups undergoing amygdala- or PTZkindling and treated or not with enalapril (10mg/kg/day; see section Drugs) in order to study strain predisposition to develop kindling and its relation to cerebral small vessels disease (CSVD). Pharmacological response to losartan or carbamazepine was only tested in groups undergoing amygdala kindling without enalapril treatment and of 16 weeks of age. Abbreviations: SHRs = Spontaneously hypertensive rats; WKYs = Wistar Kyoto rats; En = enalapril.
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Figure 2. Afterdischarge (AD) parameters before and after amygdala kindling development. A) Afterdischarge threshold determined the day before the first day of the stimulation period (prekindling; defined as the lowest electrical stimulus eliciting an AD lasting at least 3s) and at the 5th stage 5 seizure (post-kindling). B) AD duration measured on the first day of stimulation (500 μA, 1 ms, monophasic square-wave pulses, 50Hz for 1 s) and after at 5th stage 5 seizure. *significantly (p<0.05) in comparison to other age-matched groups. 5WKY = WKY of 5 weeks of age; 5WKYen = WKY of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 5SHR = SHR of 5 weeks of age; 5SHRen = SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16WKY = WKY of 16 weeks of age; 16WKYen = WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16SHR = SHR of 16 weeks of age; 16SHRen = SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s..
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Figure 3. Amygdala Kindling development. A) progression of kindling in three groups evidencing a faster development in SHRs not previously treated with enalapril (red line) in comparison to WKY control group and SHRs early treated with enalapril (10mg/kg/day) not developing cerebral small vessels disease (CSVD). B) number of afterdischarges (ADs) required for the development of the 5th stage 5 seizure. *significantly (p<0.05) in comparison to other age-matched groups. 5SHR = SHR of 5 weeks of age; 5SHRen = SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16WKY = WKY of 16 weeks of age; 16WKYen = WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16SHR = SHR of 16 weeks of age; 16SHRen = SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s..
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Figure 4: Pentylentetrazol (PTZ) Kindling development. A) progression of kindling in two groups indicating no difference between SHRs and WKY control group. B) number of PTZ administrations required for the development of the 3rd stage 5 seizure. 5SHR = SHR of 5 weeks of age; 5SHRen = SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16WKY = WKY of 16 weeks of age; 16WKYen = WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16SHR = SHR of 16 weeks of age; 16SHRen = SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s..
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Figure 5. Examples of: panels A and D) Normal histological pattern and staining for hematoxylin and eosin (H&E) and Luxol Fast Blue in subcortical structures of Wistar Kyoto rats; panels B and E) In SHR rats, note arteriolar hyalinosis (panel B, arrows) with slight spongiosis and demyelination around hyalinised vessels (panel E and enlargement E1); panels C and F) Enalapril treatment started at 30 days of age prevents the development of vascular damage (CSVD = cerebral small vessel disease). H&E images are 40x magnifications while Luxol staining images are 20x magnifications with the enlargement on panel E at 40x. Rats were aged 20 weeks on average at the time of sampling and brain sections shown in the panels were depicted from rats in the electrical kindling protocol. WKY = Wistar Kyoto rats; SHRs = Spontaneously Hypertensive rats; SHRen = SHRs treated with enalapril from 30 days of age up to the time of sampling.
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S0361-9230(17)30001-1 http://dx.doi.org/doi:10.1016/j.brainresbull.2017.02.003 BRB 9170
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
1-1-2017 30-1-2017 13-2-2017
Please cite this article as: Emilio Russo, Antonio Leo, Francesca Scicchitano, Annalidia Donato, Edoardo Ferlazzo, Sara Gasparini, Vittoria Cianci, Chiara Mignogna, Giuseppe Donato, Rita Citraro, Umberto Aguglia, Giovambattista De Sarro, Cerebral small vessel disease predisposes to temporal lobe epilepsy in spontaneously hypertensive rats, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2017.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cerebral small vessel disease predisposes to temporal lobe epilepsy in spontaneously hypertensive rats
Running Title: CSVD and Epilepsy
Emilio Russo1,*, Antonio Leo1, Francesca Scicchitano1, Annalidia Donato1, Edoardo Ferlazzo2,3, Sara Gasparini2,3, Vittoria Cianci2, Chiara Mignogna1, Giuseppe Donato1, Rita Citraro1, Umberto Aguglia2,3, Giovambattista De Sarro1
1
Science of Health Department, School of Medicine, University “Magna Graecia” of Catanzaro,
Italy; 2
Regional Epilepsy Centre, Bianchi-Melacrino-Morelli Hospital, Reggio Calabria, Italy;
3
Medical and Surgical Sciences Department, School of Medicine, Magna Græcia University of
Catanzaro, Viale Europa, Catanzaro, Italy.
*Author for correspondence: Prof. Emilio Russo Chair of Pharmacology, Department of Science of Health, School of Medicine, University of Catanzaro, Italy Via T. Campanella, 115; 88100 Catanzaro, ITALY. Phone +39 0961 3694191; Fax +39 0961 3694192; e-mail: [email protected]
Highlights
5-weeks-old spontaneously hypertensive rats (SHRs) have similar seizure susceptibility to control rats 16-weeks-old SHRs develop amygdala kindling more rapidly (less stimulations) than control rats 1
16-weeks-old SHRs display cerebral small vessels disease (CSVD) which is prevented by enalapril long-term treatment. Enalapril treatment, inhibiting the development of CSVD, normalizes amygdala kindling development in SHRs CSVD in SHRs is associated with a higher susceptibility to amygdala kindling.
Abstract The link between cerebral small vessel disease (CSVD) and epilepsy has been poorly investigated. Some reports suggest that CSVD may predispose to temporal lobe epilepsy (TLE). Aim of this study was to evaluate whether spontaneously hypertensive rats (SHRs), an established model of systemic hypertension and CSVD, have a propensity to develop TLE more than generalized seizures. To this aim, amygdala kindling, as a model of TLE, and pentylenetetrazole (PTZ)-induced kindling, as a model of generalized seizures, have been used to ascertain whether SHRs are more prone to TLE as compared to Wistar Kyoto control rats. While young SHRs (without CSVD) do not differ from their age-matched controls in both models, old SHRs (with CSVD) develop stage 5 seizures in the amygdala kindling model (TLE) faster than age-matched control rats without CSVD. At odds, no differences between old SHRs and age-matched controls was observed in the development of PTZ kindling. Enalapril pre-treatment prevented the development of CSVD and normalized kindling development to control levels in SHRs. No difference was observed in the response to pharmacological treatment with carbamazepine or losartan. Overall, our study suggests that uncontrolled hypertension leading to CSVD might represent a risk factor for TLE. Further experimental studies are needed to unravel other risk factors that, along with CSVD, may predispose to TLE.
Keywords: SHRs; stroke; hypertension; kindling; pentylenetetrazole.
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1. Introduction Experimental and clinical studies suggest that systemic hypertension and cerebral small vessel disease (CSVD) increase the risk of epilepsy even in the absence of prior clinically detected stroke [5, 17, 23, 24]. CSVD has received little attention until modern brain imaging technologies allowed to detect small deep infarcts and white matter rarefaction (i.e. leukoaraiosis) [25]. Leukoaraiosis may be observed in adults with otherwise unexplained new-onset epilepsy, but it is not clear whether leukoaraiosis represents a mere incidental radiologic finding (i.e. epiphenomenon) or plays an epileptogenic role [8, 9]. Clinical studies suggest that temporal lobe epilepsy (TLE) predominates in epileptic patients with leukoaraiosis [9, 16]. A link between TLE and alterations in the function of renin-angiotensin system (RAS) has been hypothesized [21, 22, 26]. Indeed, type 1 and 2 angiotensin receptors (AT) were found to be up-regulated in the cortex and hippocampus of patients with TLE [1]; moreover, AT-converting enzyme inhibitors (ACEi) displayed antiepileptic activity in some animal models of seizures/epilepsy [7, 19, 21]. Finally, previous reports indicate that spontaneously hypertensive rats (SHRs) could be considered as a suitable tool to study the link between hypertension and epilepsy [37, 38] and, notably, CSVD was observed in this strain [13]. Of note, SHRs display CSVD as a consequence of uncontrolled hypertension [3] and may therefore be used to study the comorbidity between hypertension, CSVD and epilepsy [29]. The primary aim of this study was to evaluate the propensity and possible differences of SHRs to develop either TLE by inducing amygdala kindling (a TLE model) or pentylenetetrazole (PTZ) kindling (a generalized seizures model) in 16-week-old SHRs and age-matched Wistar Kyoto (WKY) control rats. The secondary aims were: 1) to assess the role of ACEi to prevent hypertension-related CSVD and epilepsy by administrating enalapril before both types of kindling in the two strains; 2) to evaluate the role of hypertension or genetic factors in TLE development before CSVD onset by applying the same protocols in 5-week-old SHR and in age-matched WKY
3
control rats; 3) to test seizure response to losartan vs carbamazepine in amygdala-kindled rats of both strains.
2. Materials and Methods 2.1 Animals A total of 64 male SHRs and 64 male WKY rats of 3 weeks of age were obtained from Charles River (Milan, Italy). Rats were housed in stable conditions of humidity (60 ± 5%) and temperature (21 ± 2°C), kept under a reversed light/dark (12/12 h) cycle (light on at 19:00 h) and given free access to food and water until the time of experiments. The experimental procedures were carried out in accordance with the guidance and general recommendations with international and national law and policies (EU Directive 2010/63/EU for animal experiments, ARRIVE guidelines and the Basel declaration including the 3R concept) and after approval by the local ethical committee. Measures were included in the protocols to minimize pain and discomfort to the animals and minimize animal usage.
2.2 Drugs Pentylenetetrazole (PTZ; Sigma, Milan, Italy) was dissolved in 0.9% saline and injected s.c. in a volume of 1.0 mL/kg, at a subconvulsive dose of 30 mg/kg every other day until development of kindling or up to 8 weeks [6]. Enalapril (Germed Pharma S.p.A Cinisello Balsamo, Italy) was orally administered at a dose of 10 mg/kg/day by dissolving 20 mg in 120 ml of consumable water (calculated on the basis of the knowledge that rats drink on average 10–12 ml/100 g/day) [4]. The dose of enalapril was based on previous reports and a short preliminary experiment with two doses (10 and 20 mg/kg; n=3 per
4
group) administered for 10 days in which the highest dose appeared to be not well-tolerated following a behavioral evaluation by an expert veterinary [2]. Losartan (Sandoz; Varese, Italy), at the dose of 50 mg/kg i.p., was dissolved in 0.9% saline. Carbamazepine (Sigma-Aldrich Milan, Italy), at the dose of 50 mg/kg i.p., was dissolved in dimethyl sulfoxide (DMSO).
2.3 Surgery and electrical kindling procedure Thirty-two SHRs and 32 WKY rats were anesthetized by a tiletamine/zolazepam mixture (1:1; Zoletil 100®; 50 mg/kg i.p.; VIRBAC Srl, Milan, Italy); a bipolar electrode was implanted into the right hemisphere in the basolateral amygdala (AP= 2.2; L= 4.8; H= 8.5 in mm relative to bregma) according to Paxinos and Watson [27], as previously described [18]. All rats after surgery were randomly divided into 8 groups (n=8 for all groups; see Figure 1 for an experimental protocol scheme): 1) WKY of 5 weeks of age (5WKY); 2) WKY of 5 weeks of age treated with enalapril 10 mg/kg/day o.s. (5WKYen); 3) SHR of 5 weeks of age (5SHR); 4) SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s. (5SHRen); 5) WKY of 16 weeks of age (16WKY); 6) WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s. (16WKYen); 7) SHR of 16 weeks of age (16SHR); 8) SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s. (16SHRen). The administration of enalapril was started in all treated groups at 30 days of age and continued up to end of the experimental protocol (e.g. acquisition of kindling status). Electrical stimulation (Grass S88K stimulator) of the amygdala was started after a recovery period of at least 1 week in all groups (range 7-10 days). Afterdischarge Threshold (ADT) was determined, using stimuli of increasing intensity (20μA steps starting from 10μA) presented at 1 min intervals until an AD occurred, the day before the first day of the stimulation period and it was defined as the lowest electrical stimulus that elicited an afterdischarge (AD) lasting at least 3s [18]. From the next
5
day on, pulsed electrical stimulation (500 μA, 1 ms, monophasic square-wave pulses, 50Hz for 1 s) were delivered at intervals of 1 day until 5 consecutive fully kindled seizures (stage 5; seizure severity classified by Racine scale) were elicited [18, 30, 31]. AD duration was also measured on the first day of stimulation and after 5 stage 5 seizures. To test a putative potential difference in pharmacological response between kindled rats of the two different strains, two days after reaching a fully kindled state of 5 stage 5 seizures, the effect on the behavioral seizure score of a single dose of losartan (50 mg/kg) or carbamazepine (50 mg/kg) administered i.p. was assessed. Rats were tested at 60 min after drug administration. The doses of losartan and carbamazepine are consistent with efficacious doses of these drugs in standard kindling and other rodent seizure models [15, 20].
2.4 Pentylenetetrazole Kindling protocol Chemical kindling was induced in 32 SHRs and in 32 WKY rats by pentylenetetrazole (PTZ, 30 mg/kg s.c.) injected every other day at the most up to 8 consecutive weeks in the morning between 9:00 and 11:00 [6]. Animals were randomly divided into 8 groups (n= 8for all groups) as above described for electrical kindling and also in this case 4 out of 8 groups received enalapril (10 mg/kg/day) during the entire time-window of PTZ administration (see Figure 1). After PTZ administration, rats were placed in a Plexiglas box (100 cm wide × 100 cm long × 50 cm high) and observed for 30 min and seizures scored according to the following scale: 0 = no change in behavior; 0.5 = Abnormal behavior (sniffing, extensive washing, orientation); l = isolate myoclonic jerks; 2 = Atypical (unilateral or incomplete) clonic seizures; 3 = fully developed bilateral forelimb clonus; 3.5 = forelimb clonus with a tonic component and twist of body; 4 = Tonic-clonic seizure with suppressed tonic phase; only clonus of all limbs; 5 = fully developed tonic-clonic seizures [6, 40]. The maximum response was recorded for each animal. Rats were
6
considered fully kindled when exhibiting 3 consecutive stage 5 seizures, and then the treatment was discontinued.
2.5 Histological analysis Immediately after either electrical stimulation or PTZ administration, rats were anesthetized with a mixture of tiletamine/zolazepam (1:1; Zoletil 100®; 50 mg/kg, i.p.; VIRBAC Srl, Milan, Italy) and intracardiacally infused with cold phosphate-buffered saline (PBS, pH 7.4) and, consequently, with cold mixture of 4% paraformaldehyde (PFA), 0.2% saturated picric acid, and PBS. Brains were removed and post-fixed overnight at 4° C in the same fixative solution. After paraffin embedding, tissue sections of 4m representative of a coronal cut on bregma were obtained and sections were stained with eosin-hematoxylin for morphology and Luxol fast blue (Klüver Barrera method staining kit; Bioptica; Milan, Italy; Code: W01030799), as previously described [14, 32]. Cortical and subcortical structures were examined for signs of arteriolar hyalinosis and/or vascular damage.
2.6 Statistical analysis Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA 92037, USA). The two age subgroups (5 and 16 weeks old rats) were always analyzed separately and not compared. The ADT and AD duration within age groups was compared with a two-way (factors = strain and treatment) ANOVA for repeated measures followed by Tukey’s post hoc test. Significance of differences in scores of stage 5 seizures were compared using two-way (factors = strain and treatment) ANOVA followed by Bonferroni’s post-hoc test.
3. Results 3.1 Electrical Kindling in SHRs and WKY rats 7
ADT was not significantly different among all groups both on stimulation day 1 and after 5 stage 5 seizures (Figure 2A). AD duration was always significantly longer after kindling development and it was not significantly different between groups with the only exception of 16-week-old untreated SHRs which had a significantly (p<0.05) longer AD on the first day (ADT determination day) in comparison to all other 16-week-old groups (Figure 2B). In addition, 16-week-old untreated SHRs had a significantly faster rate of kindling development (number of AD stimulations: 14 vs 19; p < 0.0001) in comparison to age-matched WKY rats; enalapril normalized kindling development in this strain/group (Figures 3A, B). Interestingly, 5-week-old SHRs did not kindle before agematched WKY rats, and treatment did not influence outcome in all 5-week-old strains (Figure 3B). Carbamazepine reduced seizure score (from an average 5 to 3) in all 16-week-old kindled strains, while losartan had no effects (data not shown).
3.2 PTZ Kindling development Kindled seizures were directly proportional and cumulative to repeated PTZ administration in all strains. None of the animals convulsed on the first administration. No difference in seizure score was found between SHRs and controls at all ages. Both treated and untreated 16-week-old SHR had a similar rate of kindling development in comparison to age-matched WKY rats (Figures 4A, B). 5week-old SHRs did not kindle before age-matched WKY rats, and treatment did not influence kindling process (Figure 4B). In particular, the 3rd stage 5 seizure was achieved after 18 PTZ administrations in all 16-week-old strains and after 16 PTZ administrations in all 5-week-old strains (Figure 4A, B).
3.3 Histological analysis No signs of CSVD were present in all 5-week-old groups (age at analysis: 8 weeks of age in the amygdala kindling groups and 10 weeks of age in the PTZ groups). In the 16-week-old groups, untreated WKY rats did not show CSVD after both PTZ- and amygdala- kindling (age at analysis: 8
20 weeks of age in the amygdala kindling and 22 in the PTZ kindling groups: Figure 5A, D). At odds, untreated SHRs had clear signs of CSVD after both PTZ- and amygdala- kindling (age at analysis: 19 weeks of age in the amygdala kindling and 22 in the PTZ kindling groups: Figure 5B, E). CSVD was uniformly distributed in the cortex, basal ganglia, thalami and subcortical white matter. No interlobar differences in CSVD distribution were observed. Notably, no signs of CSVD were present in enalapril-treated SHRs groups after both PTZ- and amygdala- kindling (age at analysis: 20 weeks of age in the amygdala kindling and 22 in the PTZ kindling groups: Figure 5C, F).
4. Discussion We found that SHRs with hypertension-related CSVD have a higher susceptibility to TLE, as demonstrated by a significantly higher propensity to develop seizures by amygdala kindling compared to WKY control rats. Previous studies suggested that SHRs have a higher susceptibility to tonic extensor seizures [10] and TLE [11, 33, 35, 36], but the exact role of CSVD was not evaluated. We hypothesized that CSVD might play an epileptogenic role. Actually, an early treatment with enalapril prevented the development of CSVD and reduced the propensity of SHRs to develop amygdala-kindled seizures to the same levels revealed in control rats. Altogether, these findings are in keeping with human evidence showing that TLE predominates in epileptic patients with leukoaraiosis [9]. However, our neuropathological data do not clarify the pathogenic role of hypertension-related CSVD on TLE, since small vessel damage did not predominate in temporal lobes of 16-week-old SHRs. Further functional, neuropathological and molecular studies are needed to disentangle this issue. Hypertension has been suggested as an independent risk factor for epilepsy in human studies, although a specific predisposition for TLE has never been demonstrated. Ng et al. [24] found that a history of systemic hypertension represented a risk factor (OR = 1.57) for epilepsy in a case-control 9
study. Hesdorffer et al. [12] found that severe uncontrolled hypertension was associated with an 11fold increased risk of unprovoked seizures in a cross-sectional, case-control study. Finally, Li et al. [17] showed that chronic hypertension (evaluated as left ventricular hypertrophy) represented an independent risk factor (OR = 2.9) for epilepsy in a cross-sectional, case-control study. In our study, we found that only 16-week-old SHRs (with CSVD) had a higher propensity to develop seizures by amygdala kindling as compared to control rats, while 5-week-old SHRs (without CSVD) did not differ from control rats. Interestingly, upregulation of AT1 and AT2 receptors has been found in the hippocampus of patients who underwent temporal lobectomy for refractory epilepsy, suggesting the involvement of renin-angiotensin system in epileptogenesis in these patients [36]. Animal studies corroborate the evidence that an independent renin-angiotensin system exists in the central nervous system and that it may be involved in seizure susceptibility [28, 34, 36, 39]. Tchekalarova et al. [34, 39] showed that the subchronic (14 days) or long-term (16 weeks) treatment with losartan (10 mg/kg) after kainate-induced status epilepticus (SE) reduce seizure frequency and duration, and it may have a neuroprotective effect in the CA3 hippocampus area and septo temporal hilus of the dentate gyrus in SHRs. In our study, losartan was ineffective to reduce seizure severity in amygdala-kindled strains. These findings do not support an antiepileptic effect of losartan in these rat model. Moreover, a single partially effective dose of carbamazepine (50mg/kg) was used to determine whether kindled seizures in SHRs would have a possible different pharmacological responsiveness; no differences were observed, therefore, temporal lobe seizures in the two strains have a likely identical drug sensitivity. Finally, the role played by the genetic predisposition of SHRs for TLE seems to be irrelevant since 5-week-old SHRs without CSVD have not a higher susceptibility to develop amygdala kindled seizures as compared to age-matched control rats.
4.1 Conclusions 10
Overall, our study suggests that uncontrolled hypertension leading to CSVD or small vessels damage might represent a risk factor for the development of TLE more than other epilepsy types. Further studies are needed to clarify whether this result is clinically relevant and to understand which might be other risk factors that need to be combined to CSVD in order to develop TLE as suggested by previous clinical studies [8, 9, 23].
Conflict of interest Authors have nothing to declare.
Acknowledgements None
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Figure Legends
Figure 1. Scheme of the experimental protocol used in the study. SHRs and WKY rats of the two ages considered (5 and 16 weeks) were divided in two subgroups undergoing amygdala- or PTZkindling and treated or not with enalapril (10mg/kg/day; see section Drugs) in order to study strain predisposition to develop kindling and its relation to cerebral small vessels disease (CSVD). Pharmacological response to losartan or carbamazepine was only tested in groups undergoing amygdala kindling without enalapril treatment and of 16 weeks of age. Abbreviations: SHRs = Spontaneously hypertensive rats; WKYs = Wistar Kyoto rats; En = enalapril.
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Figure 2. Afterdischarge (AD) parameters before and after amygdala kindling development. A) Afterdischarge threshold determined the day before the first day of the stimulation period (prekindling; defined as the lowest electrical stimulus eliciting an AD lasting at least 3s) and at the 5th stage 5 seizure (post-kindling). B) AD duration measured on the first day of stimulation (500 μA, 1 ms, monophasic square-wave pulses, 50Hz for 1 s) and after at 5th stage 5 seizure. *significantly (p<0.05) in comparison to other age-matched groups. 5WKY = WKY of 5 weeks of age; 5WKYen = WKY of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 5SHR = SHR of 5 weeks of age; 5SHRen = SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16WKY = WKY of 16 weeks of age; 16WKYen = WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16SHR = SHR of 16 weeks of age; 16SHRen = SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s..
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Figure 3. Amygdala Kindling development. A) progression of kindling in three groups evidencing a faster development in SHRs not previously treated with enalapril (red line) in comparison to WKY control group and SHRs early treated with enalapril (10mg/kg/day) not developing cerebral small vessels disease (CSVD). B) number of afterdischarges (ADs) required for the development of the 5th stage 5 seizure. *significantly (p<0.05) in comparison to other age-matched groups. 5SHR = SHR of 5 weeks of age; 5SHRen = SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16WKY = WKY of 16 weeks of age; 16WKYen = WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16SHR = SHR of 16 weeks of age; 16SHRen = SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s..
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Figure 4: Pentylentetrazol (PTZ) Kindling development. A) progression of kindling in two groups indicating no difference between SHRs and WKY control group. B) number of PTZ administrations required for the development of the 3rd stage 5 seizure. 5SHR = SHR of 5 weeks of age; 5SHRen = SHR of 5 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16WKY = WKY of 16 weeks of age; 16WKYen = WKY of 16 weeks of age treated with enalapril 10 mg/kg/day o.s.; 16SHR = SHR of 16 weeks of age; 16SHRen = SHR of 16 weeks of age treated with enalapril 10 mg/kg/day o.s..
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Figure 5. Examples of: panels A and D) Normal histological pattern and staining for hematoxylin and eosin (H&E) and Luxol Fast Blue in subcortical structures of Wistar Kyoto rats; panels B and E) In SHR rats, note arteriolar hyalinosis (panel B, arrows) with slight spongiosis and demyelination around hyalinised vessels (panel E and enlargement E1); panels C and F) Enalapril treatment started at 30 days of age prevents the development of vascular damage (CSVD = cerebral small vessel disease). H&E images are 40x magnifications while Luxol staining images are 20x magnifications with the enlargement on panel E at 40x. Rats were aged 20 weeks on average at the time of sampling and brain sections shown in the panels were depicted from rats in the electrical kindling protocol. WKY = Wistar Kyoto rats; SHRs = Spontaneously Hypertensive rats; SHRen = SHRs treated with enalapril from 30 days of age up to the time of sampling.
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