Carnosine, a precursor of histidine, ameliorates pentylenetetrazole-induced kindled seizures in rat.

Abstract

Carnosine (beta-alanyl-l-histidine) has been characterized as a putative neurotransmitter. However, so far, understanding of the role of carnosine in the brain is very limited. The objective of this study was to examine the effects of carnosine on the development of pentylenetetrazol (PTZ) kindling seizures and protection against the PTZ kindled seizures in rats. Chemical kindling was elicited by repeated intraperitoneal injection of PTZ (35 mg/kg) once every 48 h until the occurrence of Stage 4-5 seizures, and the seizure activity of kindling was recorded for 30 min. In an acute PTZ challenge study, 60 mg/kg PTZ was used to induce kindled seizure. Injection of carnosine (200, 500 mg/kg, i.p.) significantly decreased seizure stage, and prolonged the latencies for myoclonic jerks, in a dose- and time-dependent manner. In the seizure development process, 500 mg/kg carnosine also significantly delayed the onset of PTZ kindled seizures. In addition, carnosine significantly reversed decreased histamine levels induced by PTZ kindled seizure in the hippocampus. These results indicate that carnosine can protect against PTZ-induced seizures in both the development of kindling and the challenge process in rats. The results suggest that carnosine might be an endogenous anticonvulsant factor in the brain and can be used as a new antiepileptic drug in future.

Full text

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Neuroscience Letters xxx (2006) xxx–xxx
Carnosine, a precursor of histidine, ameliorates pentylenetetrazole-induced
kindled seizures in rat
Xiao-hua Wua,b,c, Mei-ping Dingb, Zheng-Bing Zhu-Gea, Yuan-Yuan Zhua,
Chun-lei Jina, Zhong Chena,∗
aDepartment of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310031, China
bDepartment of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China
cDepartment of Neurosurgery, Jinhua Central hospital, Jinhua 321000, China
Received 14 September 2005; received in revised form 10 February 2006; accepted 12 February 2006
Abstract
Carnosine (?-alanyl-l-histidine) has been characterized as a putative neurotransmitter. However, so far, understanding of the role of carnosine
in the brain is very limited. The objective of this study was to examine the effects of carnosine on the development of pentylenetetrazol (PTZ)
kindling seizures and protection against the PTZ kindled seizures in rats. Chemical kindling was elicited by repeated intraperitoneal injection of
PTZ (35mg/kg) once every 48h until the occurrence of Stage 4–5 seizures, and the seizure activity of kindling was recorded for 30min. In an acute
PTZchallengestudy,60mg/kgPTZwasusedtoinducekindledseizure.Injectionofcarnosine(200,500mg/kg,i.p.)significantlydecreasedseizure
stage, and prolonged the latencies for myoclonic jerks, in a dose- and time-dependent manner. In the seizure development process, 500mg/kg
carnosine also significantly delayed the onset of PTZ kindled seizures. In addition, carnosine significantly reversed decreased histamine levels
induced by PTZ kindled seizure in the hippocampus. These results indicate that carnosine can protect against PTZ-induced seizures in both the
development of kindling and the challenge process in rats. The results suggest that carnosine might be an endogenous anticonvulsant factor in the
brain and can be used as a new antiepileptic drug in future.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Carnosine; Pentylenetetrazol kindling; Seizure; Histamine
The role of brain histamine in regulating seizure susceptibil-
ity has recently been documented, and an anticonvulsant action
of endogenous histamine has been postulated [5,14,19,23,28].
Both histamine and histidine increase the threshold for amyg-
daloid kindling and pentylenetetrazol (PTZ)-induced seizures
[4,14,27]. Several H1receptor antagonists, such as diphenhy-
dramineandchlorpheniramine,occasionallyinduceconvulsions
in epileptic patients, healthy children and rodents [13,15,26].
We also reported that the seizure development induced by
PTZ is facilitated in H1receptor knockout mice and histidine
decarboxylase-deficient mice compared with wild-type mice
[5].
However, histamine cannot cross the blood–brain barrier
(BBB) and may be involved in brain inflammation [21]. The
∗Correspondingauthorat:DepartmentofPharmacology,SchoolofMedicine,
Zhejiang University, Hangzhou 310031, China. Tel.: +86 571 87217446;
fax: +86 571 87217446.
E-mail address: chenzhong@zju.edu.cn (Z. Chen).
infusion of histamine into rat substantia nigra results in an
acute inflammatory response manifested by a loss of glial fibril-
lary acidic protein-immunolabeled astrocytes [24]. On the other
hand, carnosine has been characterized as a putative neurotrans-
mitter in olfactory receptor neurons [3]. Carnosine may be a
potentprotectiveagentintheCNS[2,11,22]andmayplayarole
in neuron–glia interactions [16]. However, little is known about
theroleofcarnosineinthebrain.Carnosineservesasareservoir
for histidine, which is a precursor of histamine [8]. Given the
relationshipbetweencarnosineandhistamine,itisproposedthat
carnosine may be a new histaminergic drug and could be used
clinically instead of histamine. We have recently reported that
carnosine can prevent amygdaloid kindling seizures in rats [13].
Yet, there is limited information about the effects of carnosine
on PTZ-induced kindling seizures, which is an animal model
of human absence epilepsy and myoclonic, generalized tonic-
clonic seizures. Therefore, our investigations were designed
to elucidate the pharmacological mechanisms of the effects of
carnosine on both the development of kindling seizures and the
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2006.02.031
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X.-h. Wu et al. / Neuroscience Letters xxx (2006) xxx–xxx
kindled seizures themselves following PTZ administration in
rats.
All experiments were carried out in accordance with the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals. The animals used in this study were male
Sprague–Dawley rats (220–300g, Grade II, Certificate No. 22-
9601018, Experimental Animal Center, Zhejiang University),
maintained in individual cages with a 12-h light–dark cycle
(lights on from 8:00 to 20:00). Food and water were given ad
libitum. Experiments were carried out each day between 12:00
and 17:00.
To induce kindling, a 35mg/kg dose of PTZ (Sigma, St.
Louis, MO, USA) was injected i.p. every 48h [4,9,27]. After
eachPTZtreatment,ratswereplacedseparatelyunderglassfun-
nels, the appearance of clonic and tonic seizures were recorded
during individual observation for 30min. Seizure intensities
wereclassifiedasfollows:0:noresponse;Stage1:earandfacial
twitching;Stage2:convulsivewavesthroughoutthebody;Stage
3: myoclonic jerks, rearing; Stage 4: turning over onto one side;
Stage 5: turning over onto the back, generalized tonic-clonic
seizures. In addition, the latency to the onset of myoclonic jerks
was measured and analyzed. In an acute PTZ challenge study,
60mg/kg PTZ was used to elicit kindled seizure. Each rat was
treated by one dose of PTZ, and also measures for 30min.
Carnosine was injected i.p. 2h before PTZ treatment. Thirty
min after PTZ acute treatment, rats were quickly sacrificed by
decapitation. The brain was removed and placed on an ice-
cold stainless steel plate, and subsequently dissected into cortex
and hippocampus. The brain tissues were stored at −80◦C
until assayed. The brain tissue was homogenized (Polytron
homogenizer, Kinematica, Lucern, Switzerland) in 3% perchlo-
ric acid containing 5mM disodium EDTA and 5-hydro-N?-
methyltryptamineandthencentrifugedat15,000×gfor20min
at 4◦C. The tissue samples were analyzed by high-performance
liquid chromatography (HPLC) combined with electrochemical
detectionusingatechniquedevelopedinourlaboratory[13].The
system consisted of a model 582 pump, a model 540 autosam-
pler and a 4-channel CoulArray electrochemical detector (ESA,
Chelmsford,MA,USA).TheHPLCwascontrolledandthedata
acquired and analyzed using CoulArray®software. After react-
ing with the derivate o-phthalaldehyde, analytes were separated
on a 3?m, 3mm×50mm Capcell Pak MG C18 column (Shi-
seido,Tokyo,Japan).Atwo-componentgradientelutionsystem
wasused,withcomponentAofthemobilephasebeing100mM
Na2HPO4, 13% acetonitrile, and 22% methanol, pH 6.8, and
component B being similar to A except with 5.6% acetonitrile
and 9.4% methanol. A gradient elution profile was used as fol-
lows: 0–3.5min, isocratic 100% B; 3.5–20min, linear ramp to
0% B; 20–22min, isocratic 0% B; 22–23min, linear ramp to
100% B; 23–30min, isocratic 100% B. The flow rate was set to
0.75mL/min. The temperature of the column was maintained at
38◦C. The first cell was set at +250mV, the second at +350mV.
The detection limit (signal/noise ≥3) was 1ng for histamine.
All results are expressed as the mean±S.E.M. Statistical
analyses used SPSS 11.5 for Windows. One-way analysis of
variance(ANOVA)withDunnett’stestwasusedtocalculatethe
significance. In the case of kindling seizure development, the
Fig. 1. Effect of carnosine on seizure development of PTZ (35mg/kg) kindling
in rats. Carnosine was injected i.p. 2h before PTZ treatment. (A) Effect on
seizure stage. Open circles indicate saline-treated group; closed circles indicate
carnosine(500mg.kg)treatedgroup.(B)Effectonlatencytoonsetofmyoclonic
jerks.Openbars:saline-treatedgroup.Filledbars:carnosine-treatedgroup.Each
value represents the mean±S.E.M. from 11 to 14 rats.*p<0.05 compared to
control group.
Kruskal–Wallis test followed by multiple comparison (Bonfer-
roni correction) was used to calculate significance.
Carnosine inhibited PTZ-induced seizure development
(p<0.05, Fig. 1). At a dose of 500mg/kg it decreased PTZ-
induced seizure stage, and prolonged the latency for myoclonic
jerks (p<0.05). In the acute PTZ challenge process, as shown
in Fig. 2, carnosine also decreased the stage of PTZ kindled
seizures dose- and time-dependently. At 100mg/kg, it slightly
inhibited the PTZ kindled seizures, but the effect did not reach
significance. Carnosine at 200mg/kg decreased seizure stage
and prolonged the latencies for myoclonic jerks (p<0.05) at
2h after injection. At 500mg/kg it inhibited the PTZ kindled
seizures at 1 and 2h after injection (p<0.05). At 1000mg/kg,
significant effects on PTZ-induced seizures were not observed,
resulting in a bell-shaped dose response curve. In addition, as
shownintheFig.3,PTZkindledseizuresignificantlydecreased
histamine levels both in the cortex and hippocampus. Carnosine
(500mg/kg) slightly increased the histamine level in the cortex
and significantly increased histamine levels in the hippocampus
(p<0.05), at 1000mg/kg, it significantly increased histamine
levels both in the cortex and hippocampus (p<0.05, Fig. 3).
In the present study, we provide the first evidence that
carnosine significantly protected against both the development
and process of PTZ-induced kindled seizures. Previous data
showed that long-term treatment with a low histamine diet

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X.-h. Wu et al. / Neuroscience Letters xxx (2006) xxx–xxx
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Fig. 2. Dose- and time-dependent effects of carnosine on PTZ (60mg/kg) kindled seizures in rats. Carnosine was injected i.p. 2h before PTZ acute treatment. (A)
Effectsonseizurestage.(B)Effectsonlatencytoonsetofmyoclonicjerks.Thecarnosine-treatedgroupsweregivendosesof100(closedcircles),200(opentriangles)
or 500mg/kg (closed triangles). Controls were given saline (open circles). Each value represents the mean±S.E.M. from 11 to 14 rats.*p<0.05 compared to control
group.
enhances seizure development induced by PTZ kindling [12],
while this effect can be reversed by simultaneous treatment
with a high carnosine diet (data not shown). Because carno-
sine can easily cross the BBB and has few side effects [10], it
may be a new potential anticonvulsant drug for clinical therapy
of human absence epilepsy and myoclonic, generalized tonic-
clonic seizures.
Our previous data showed that carnosine at doses of 500,
1000 and 1500mg/kg significantly decreases seizure stage and
afterdischarge duration in amygdaloid kindled seizures, in a
dose-dependent and time-related manner (peaking at 1h), and
increases hippocampal histamine levels, which also peak at 1h
[13].However,inthepresentstudy,carnosineatthelowerdoses
of 200 and 500mg/kg protected against PTZ kindled seizures
with a peak at 2h. The inhibition showed a bell-shaped curve,
decreasingatthehigherdosesof1000mg/kgatwhichcarnosine
markedly increased histamine levels both in the cortex and hip-
pocampus. It is hard to explain why it produced a bell-shaped
effect. Wada et al. also reported histidine can suppress kindled
seizure generalization, whereas it accelerates the development
of kindling, suggesting a biphasic action of histamine in the
kindling model [25]. This may be due to different mechanisms
underlying the different seizures. Further studies are needed,
therefore, to examine the bell-shaped curve of carnosine effects
in the kindling model. These findings at least suggest that dif-
ferent doses of carnosine may contribute to the treatment of
different types of epilepsy.
It was interested to find that an acute PTZ treatment signif-
icantly decreased histamine levels both in the cortex (33.5%)
and hippocampus (29.4%), and in contrast that carnosine
(500mg/kg, a dose that inhibited PTZ kindled seizures) simul-
taneously caused a marked increase of histamine levels in the
hippocampus. However, cortical levels of histamine did not sig-
nificantlychange.Previousdataalsoshowedthatcarnosineonly
causes a marked increase of histamine levels in the hippocam-
pus and amygdala but not cortex [13]. This finding suggests
that the carnosine-induced antiseizure action is dependent on an
increase in histamine levels in the hippocampus, although it is
wellknownthatthecortexplaysanimportantroleinelectrically
kindled seizures. For example, we have also reported that once
fullykindledbyPTZ,thistypeofseizureissensitivetohistamine
levelsinthecortex[13].Wehavenofurtherdatatoexplainthese
phenomena. In addition, it is reported that carnosine inhibits
the activity of histidine decarboxylase partially purified from
whole bodies of fetal rats [18], which suggest that carnosine
maydecreasehistaminelevel.However,carnosineincreasedhis-
tamine level in the hippocampus after PTZ acute treatment. It
may be that the activity of histidine decarboxylase influenced
by carnosine may be brain region-dependent; secondary that,
the stress of PTZ acute treatment may change the enzyme sen-
Fig. 3. Effects of carnosine on histamine levels in cortex and hippocampus of rats. Carnosine was injected i.p. 2h before PTZ treatment. Thirty minutes after PTZ
acute treatment, rats were quickly sacrificed by decapitation. Each value represents the mean±S.E.M. of 8–10 rats.*p<0.05 compared to control group.*p<0.05
compared to PTZ group.

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X.-h. Wu et al. / Neuroscience Letters xxx (2006) xxx–xxx
sitivity to carnosine. Therefore, the observed effect of carnosine
is, at least in part, due to increased histamine synthesis in the
hippocampus. Thus, these results provide additional evidences
to support the idea that carnosine can be metabolically trans-
formed into histamine in the CNS. Further biochemical studies
are needed to investigate the mechanism.
To date, carnosine has been proposed to provide a non-
mast cell source of histidine in many histamine-rich tissues,
available for histamine synthesis during periods of physiolog-
ical stress [6]. Carnosine (via histidine) may be metabolically
transformed into histamine [7,10]. Evidence of inflammatory
processes in the clinical manifestations and neuropathologi-
cal sequelae of epilepsy have accumulated in the last decade
[17]. So, it may be necessary to simultaneously provide anti-
inflammatory treatment when carrying out anticonvulsant ther-
apy.Carnosineisapowerfulanti-inflammatory,presumablydue
to its anti-oxidant and anti-glycation properties [1,20]. We also
foundthatchronictreatmentwithcarnosinecandecreaseTNF-?
in the hippocampus (data not shown). Therefore, the remark-
able anti-inflammatory and anticonvulsant effects of carnosine
reflect the high potential therapeutic value of this compound as
an antiepileptic drug.
In summary, our experiments provide preliminary evidence
that carnosine has a significant anticonvulsant effect on PTZ-
kindled seizures in rats. The present study indicates that carno-
sinemaybeanendogenousanticonvulsantfactorinthebrainand
calls for its further study as a potentially efficient antiepileptic
drug.
Acknowledgements
This Project was supported by grants from the National
Natural Science Foundation of China (30371638, 30472013),
the Zhejiang Provincial Natural Science Foundation of China
(R303779) and the Zhejiang Provincial Scientific Research
Foundations (2004C34002). We are very grateful to Dr. Iain
C Bruce (Hongkong Univ) for his help in the correction of the
language.
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