Effect of Lithium Chloride Concentration on Its Neuroprotective Properties in Ischemic Stroke in Rats

Для цитирования: Р. А. Черпаков, О. А. Гребенчиков. Влияние концентрации хлорида лития на его нейропротекторные свойства при ишемическом инсульте у крыс. Общая реаниматология. 2021; 17 (5): 101–110. https://doi.org/10.15360/1813-9779-2021-5-101-110 [На русск. и англ.] For citation: Rostislav A. Cherpakov, Oleg A. Grebenchikov. Effect of lithium chloride concentration on its neuroprotective properties in ischemic stroke in rats. Obshchaya Reanimatologiya = General Reanimatology. 2021; 17 (5): 101–110. https://doi.org/10.15360/1813-9779-2021-5-101-110 [In Russ. and Engl.]


Introduction
Of all the diseases of the cardiovascular system, stroke attracts particular attention. This is due to both high mortality in this disease [1] and persistent disability even in case of timely medical care [2,3]. In addition, there is an increase in the incidence of this disease, in both young and the elderly population [4]. And if for persons over 65 years old the increase in rates of strokes is primarily related to the rise in life expectancy and average age in developed and developing countries [5], several factors can be identified when estimating the prevalence of this disease in the young age population [6]. Behavioral (low physical activity, smoking, alcohol and substance abuse) and agerelated (pregnancy/postpartum period, taking oral contraceptives) ones are considered to be the main factors.
Among the drugs with a strong neuroprotective effect, lithium carbonate, which has been successfully used for more than 60 years for such diseases as bipolar disorder, is particularly noteworthy [12,13]. The neuroprotective effects of lithium salts revealed in clinical practice have been confirmed in recent experimental studies both in vitro and in vivo (models of cognitive dysfunction in rats) [14,15]. The work of Ming  et al. [16], who showed for the first time the effect of various doses of lithium chloride on ischemic stroke volume, is particularly relevant. In this study a model of focal cerebral ischemia after 1 hour long middle cerebral artery occlusion with subsequent reperfusion was used. Lithium chloride was administered in doses ranging from 0.5 mEq/kg (~21 mg/kg) to 3 mEq/kg (~127 mg/kg) immediately after reperfusion, followed by humane euthanasia 23 hours after recanalization of the damaged brain area. The volume of brain lesions was assessed by staining with 2% 2,3,5-triphenyltetrazolium chloride. In the control group, the stroke volume was 290±12.5 mm3, and the administration of even lowest of the studied doses significantly reduced the extent of brain damage. At a dose of 21 mg/kg, the stroke volume 24 hours post stroke was 210±14.5 mm3, which was significantly lower than in the control group (P 0.01). In the groups receiving 42 mg/kg, 84 mg/kg, and 127 mg/kg, stroke area was similarly significantly lower than in the control group (P 0.001). When studying the effects of lithium chloride in models of global cerebral ischemia [17] and hemorrhagic stroke [18] its neuroprotective effects were also clearly shown.
However, in earlier studies, the extent of damage was assessed postmortem in an acute experiment, which did not provide a complete view of the effect of lithium chloride on the evolution of stroke focus and perifocal edema, and also did not allow to estimate the delayed protective effects. Moreover, in 2003 [16], the 127 mg/kg dose which is nearly 4 times higher than the maximal tolerance dose for human use proved to be the most effective in terms of neuroprotection [19]. The «narrow therapeutic window» of lithium salts is well known, that is why it is so important, knowing their distinct dose-dependent effect, to determine its optimal concentration in blood plasma for neuroprotective properties on the one hand and to avoid toxic effects on the other hand.
Therefore, the aim of our study was to investigate the effect of various concentrations of lithium chloride on the volume of ischemic stroke and perifocal edema in rats post cerebral ischemia.
Only animals that survived for 7 days were included in the final evaluation. Animals that died during the experiment were not included in the analysis of brain damage volume and were excluded from the group.
At all stages, animals were labeled by marking the base of the tail with a permanent marker. In accordance with the marking, each animal included in the study was assigned an appropriate number.
The animals were divided into 5 groups. Group 1 (n=7, mean weight 302.5±12.3 g) included sham-operated animals, which underwent a midline skin incision along the trachea projection line and whose artery was isolated as a surgical intervention with subsequent layered closure of the wound and antiseptic treatment. Group 2 was a control group (n=7, mean weight 306±11.2 g); the animals underwent focal cerebral ischemia by occluding the middle cerebral artery. Immediately after cessation of ischemia, the animals received intravenous 0.9% NaCl solution 1.5 ml/kg. Group 3 (n=7, mean weight 304±12.7 g) animals immediately after ischemia cessation animals received intravenous lithium chloride at a dose of 4.2 mg/kg, followed by the same dose of drug every 24 hours. Group 4 (n=7, mean weight 305±11.5 g) animals after removal of vascular clamp had lithium chloride 21 mg/kg injection, followed by the same dose of drug every 24 hours. Group 5 (n=7, mean weight 309±10.5 g) animals received intravenous lithium chloride, 63 mg/kg, immediately after cessation of focal ischemia and later once every 24 hours for 7 days.
The dosages of lithium chloride used were selected based on the available data on acute and chronic toxicity of the drug [13].
Induction anesthesia was performed by injecting 12% chloral hydrate solution 300 mg/kg into the peritoneum. The surgical field was sterilized with 0.05% chlorhexidine solution. After that, a midline incision was made in the neck, and the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated from the right side. The CCA was clamped with a vascular clip, and a 3-0 Vicryl ligature was placed to the ICA. The ECA was cut with scissors 3-5 mm from the bifurcation. A 0.25 mm nylon thread coated with silicone and treated with heparin solution was inserted through a section of ECA into ICA to the depth of 19-21 mm (until the MCA was occluded) and fixed with a vascular clip (MCA occlusion). The blood flow was blocked for 60 min, then the suture was extracted, which restored the blood supply in the MCA region. After that, the ECA stump was coagulated to complete tightness and the vascular clips were subsequently removed. At the end of the operation, the incision was sutured with 4-0 Vicryl and treated with 5% brilliant green dye. After the operation, body temperature monitoring was performed aimed at maintaining physiological values using infrared lamps until independent thermoregulation was restored in the animal. During the operation the body temperature was maintained at 37±0.5°C using an electric heating pad. The av-крысам вводили хлорид лития в дозе 21 мг/кг с последующим введением указанной дозы раз в 24 часа. Группа V (n=7, средняя масса 309±10,5 г) -животным сразу после прекращения фокальной ишемии, и далее в течение 7 суток один раз в 24 часа вводили хлорид лития в дозе 63 мг/кг. Применяемые дозировки хлорида лития были подобраны на основании имеющихся данных об острой и хронической токсичности препарата [13].
Normal saline in the control group and lithium chloride in groups 3, 4 and 5 were administered intravenously immediately after the vascular clips were removed. On the following days of the experiment the drug was administered once a day until euthanasia.
The extent of brain tissue damage was assessed by magnetic resonance imaging (on day 2) and by scanning the brain sections stained with 2,3,5-triphenyltetrazolium chloride (TTC-method) after euthanasia (on day 7).
The laboratory animals were sedated by inhalation of 1.5-2.0 vol% Isoflurane before placement in the MRI machine. The animals were then placed in a positioning device with a stereotaxis and thermoregulation system. All pulse sequences were pre-tested and optimized on dummies. A linear transmitter with an internal diameter of 72 mm was used to transmit the radiofrequency (RF) signal, and a surface receiving coil for the rat brain was used to detect the RF signal. The following pulse sequences (PS) were used: RARE -spin echo-based PS with TR 6000 ms, TE 63.9 ms, slice thickness 0.5 mm, matrix size 256 384, resolution 0.164 0.164 mm/pixel. Total scanning time per animal was about 25 minutes.
On day 7 after ischemia, animals were given an increased dose of chloral hydrate and decapitated. The brain was removed from the cranium and rinsed in normal saline, then cut into 2-mm-thick coronal sections using a special mold with grooves for precise cutting. Sections were stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC, «Sigma») solution in buffer with pH 7.4 for 10 minutes at 37˚C and fixed in 10% formalin solution in buffer. To obtain images, the sections were scanned from both sides on an HP Scanjet 4500c scanner (2400 dpi resolution).
Digital images of brain sections stained with TTC or T2-weighted magnetic resonance images (MR images) were used for morphometric assessment of the ischemic focus area and the volume of ischemic injury. Digital images were analyzed using ImageJ (NIH, Bethesda, USA) medical image analysis freeware. Separate measurements of total infarct area, cortex, and subcortical structures were used for each section. Areas not stained with TTC were considered to be necrotic. The 2,3,5-triphenyltetrazolium chloride itself is white, but under the action of cellular dehydrogenases it turns red due to reduction, which clearly delineates the dead tissue zone.
The volume of ischemic infarction (V) was determined using the formula: V= A d where V -volume; A -sum of the areas of all MR-sections; d -width of sections.
Also, percentage of cerebral edema was determined using the formula:

% cerebral edema = ((Sih -Sch) / Sch) 100
where Sih -area of the ipsilateral hemisphere, Sch -area of the contralateral hemisphere. The volume of ischemic lesion for each group was normalized to the mean value of infarct volume in the MCA occlusion + normal saline group.

Results and Discussion
The MRI performed on day 2 after ischemia episode showed that the stroke volume significantly differed from the control group only when lithium chloride was administered at doses of 21 mg/kg and 63 mg/kg.
The results obtained when assessing the volume of brain damage on day 2 are presented in Table 1. Stroke volume in the 4.2 mg/kg group was 7.8% lower than in the control group, and perifocal edema volume was 5.7% lower. In the group receiving the drug at 21 mg/kg, the stroke volume was reduced by 25.5% and perifocal edema by 18.6%. In the group receiving lithium chloride at a dose of 63 mg/kg, the stroke volume was reduced by 43.5% and perifocal edema by 35.4%. The brain lesion areas on MRI on day 2 are shown in Figure. Lithium chloride at doses of 21 mg/kg and 63 mg/kg significantly reduced the volume of the damage, whereas no significant difference was observed, when a dose of 4.2 mg/kg was administered.
The results obtained when assessing the stroke volume on day 7 are presented in Table 1.
In animals receiving lithium chloride at a dose of 4.2 mg/kg, the damaged area was 5.7% smaller than in the control group. The volume of damaged tissue was 21% less with the dose of 21 mg/kg and 41% less when the the dose of 63 mg/kg was used.
When determining the concentration of lithium chloride in the blood of animals, we found that the dose of 63 mg/kg was associated with the borderline toxic blood level (2.5 mmol/L) 5 minutes after administration. However, as early as one hour later, the blood concentration of the drug decreased again to a safe level. Separately, it should  Table 1. Volume of brain damage zones on day 2 and day 7 after stroke at various doses of lithium chloride, Q2 (Q1-Q3).
Note. * Differences versus controls are significant (Mann-Whitney U-test) at P 0.05.
В ходе эксперимента отдельно оценивали летальность в группах, однако ее анализ не be noted that we decided to avoid using lithium chloride at a dose of 84 mg/kg due to exceeding the threshold toxic concentration both after 5 minutes and after 1 hour.
During the experiment, mortality was evaluated separately in the groups, but its analysis was not a priority of our study. In the control group the mortality rate was 58.8% (10 out of 17 animals died). In group 3, 9 animals out of 16 died (56.25%, χ 2 =0.022, P=0.882 versus the control group). The mortality rate in group 4 was 36.3% (4 out of 11 animals died, χ 2 =1.348, P=0.246 versus the control group), in group 5 it was minimal and reached 30% (3 out of 10 animals died, χ 2 =2.095, P=0.148 versus the control group).
Currently, the molecular mechanisms of the neuroprotective effect of lithium salts are well known [10]. The first mechanism is the direct inhibition of glycogen synthase kinase 3beta (GSC-3β), the main enzyme in ischemic and pharmacological preconditioning [21]. The second mechanism is inactivation of NMDA-receptors, which leads to decrease of pro-apoptotic protein p53 activity and increase in anti-apoptotic proteins Bcl2 activity [13]. The third mechanism is the activation of PI3K/Akt signaling pathway responsible for cell survival [22].
The multifaceted mechanism of realization of the neuroprotective effect of lithium salts suggests that its use will improve the results of treatment of ischemic stroke. Moreover, there is a considerable clinical experience with lithium carbonate use. Administration of lithium carbonate in bipolar disorders has allowed to estimate its effect on stroke incidence in this group of patients, which, Whitney-according to available data, is much higher than in other populations [23]. When compared with patients on standard therapy based on antipsychotics and antidepressants, the incidence of stroke among those receiving lithium carbonate therapy was significantly lower. According to studies, with standard therapy, stroke developed in 5.4% of patients, while in those on lithium carbonate its incidence did not exceed 2.8%, which was significantly lower (P 0.05) [24,25]. Also, Mohammadianinejad S. E. [26] and Sun Y. R. [27] have shown improvement of motor functions in post stroke patients administered with lithium carbonate. Таблица 2. Концентрация хлорида лития (ммоль/л) в крови после однократного введения, Q2 (Q1-Q3). Table 2. Blood lithium chloride concentration (mmol/L) after a single injection of the drug, Q2 (Q1-Q3). Примечание. In blood at different periods after injection -в крови в разное время после введения. значимо (p 0,05) меньше. [24,25]. Так же в работах S. E. Mohammadianinejad [26] и Y. R. Sun [27] было отмечено улучшение моторных функций у пациентов, перенесших инсульт и принимавших после этого карбонат лития.

Conclusion
The results have led us to two conclusions. First, lithium chloride has a neuroprotective effect both in the early (day 2) and in the delayed (day 7) poststroke period, significantly reducing the size of the stroke and perifocal edema. Secondly, this effect is dose-dependent, being fully manifested at doses of 21 mg/kg and 63 mg/kg and having no effect on the brain lesion volume when administered at a dose of 4.2 mg/kg. At the same time, when comparing with the previously studied plasma concentrations [16], the neuroprotective effect of lithium chloride was shown to develop without exceeding the toxic concentration. Moreover, in earlier studies [23,28,29], the optimal doses for implementation of cardioand nephroprotective effect were 30 mg/kg. The «narrow therapeutic window» of lithium salts is well known, so in view of their distinct dose-dependent effect it is important to determine the optimal plasma concentration of lithium salts to implement their neuroprotective properties on the one hand and prevent toxic effects on the other hand.
The newly obtained knowledge warrants the prospects for developing a novel drug formulation (intravenous lithium chloride) for the treatment of cerebrovascular accidents.