The protective effect of L-carnitine against hippocampal damage due to experimental formaldehyde intoxication in rats E Ozmen1, SY Ozsoy2, N Donmez3, B Ozsoy4, N Yumus˛ak5 1Department of Anatomy, Faculty of Veterinary Medicine, University of Mustafa Kemal, Hatay, 2Department of Pathology, Faculty of Veterinary Medicine, University of Mustafa Kemal, Hatay, 3Department of Physiology Faculty of Veterinary Medicine, University of Selcuk, Konya, 4Department of Animal Nutrition and Diseases, Faculty of Veterinary Medicine Mustafa Kemal University, Hatay, and 5Department of Pathology, and Faculty of Veterinary Medicine, University of Harran, Sanliurfa, Turkey

Accepted October 11, 2013

Abstract We investigated the protective effects of L-carnitine on hippocampus tissue damage in rats during experimental formaldehyde (FA) intoxication. Male Wistar albino rats were assigned into four groups: (1) control (C), (2) formaldehyde (FA), (3) formaldehyde  0.5 g/kg of L-carnitine (FA  0.5 LC) (4) formaldehyde  1 g/kg L-carnitine (FA  1 LC). At the end of the 14 day trial period, animals were sacrificed by decapitation under anesthesia. The hippocampus tissue samples were extracted to measure MDA, GSH and SOD activity. Neuronal degeneration was assessed based on histopathological (hematoxylin and eosin) and immunohistochemical (anti-ubiquitin) examination. To detect oxidative stress, specimens were reacted with anti-Cu/Zn-SOD antibody. After administering L-carnitine with FA to the animals, the activities of SOD and GSH increased, but the levels of MDA decreased in hippocampus tissue. Neuronal degeneration was observed in the FA group. L-carnitine administration reduced neuronal degeneration and histological structure was similar to controls. After FA application, degenerated hippocampus neurons were stained with anti-ubiquitin and Cu/Zn-SOD antibodies; weakly positive staining was observed in Lcarnitine-treated groups. L-carnitine may be useful for preventing oxidative damage in the hippocampus tissue due to formaldehyde intoxication. Key words: formaldehyde intoxication, hippocampus, histopathology, immunohistochemistry, L-carnitine, oxidative damage, rat Formaldehyde (FA) is widely used in many industries. It is used for preservation of cadavers and fixation of tissues for histology and pathology (Smith 1992). The American Occupational Safety and Health Administration has enumerated 52 occupational groups that are exposed to FA (Dowel 1985); anatomists are among the most affected (Stroup et al. 1986). The hippocampus is part of cerebral cortex. It is a “C-shaped” structure located along the Correspondence: Dr. Sule Yurdagul Ozsoy, Faculty of Veterinary Medicine, Department of Pathology, Mustafa Kemal University, 31000, Hatay, Turkey. Tel:  90 326 2455845/1540, Fax:  90 326 2455704. E-mail: [email protected] © 2014 The Biological Stain Commission Biotechnic & Histochemistry 2014, 89(5): 336–341.


inferior horn of the lateral ventricle. The hippocampus is part of the limbic system that receives sensory fibers from several regions of the brain. It transmits information to the hypothalamus, thalamus and septal area by way of fornix. (Arıncı and Elhan 2001, Songur et al. 2001). The hippocampus plays an important role in learning and memory, data storage, and for tramsferring short-term memory to long term memory. When the hippocampus is damaged, deficits may occur in any or all of these functions. (Songur et al. 2001). Earlier studies have demonstrated the neurotoxic effects of FA in rats include behavioral disorders, mental imbalance and learning disorders (Pitten et al. 2000). Acetyl-L-carnitine occurs naturally in a variety of animal products and is available as a nutritional 336

supplement (Cos˛kun 1999, 2003). Although animal based foods contain high levels of carnitine, it is present in small amounts in plants (Carroll and Core 2001). Supplementation with acetyl-Lcarnitine may exert protective effects on the brain and peripheral nervous system. It has been reported that acetyl-L-c can be transported across membranes and can provide acetyl groups for regenerating acetyl-co-A. Acetyl-L-carnitine also can reverse the loss of neurons in certain areas of the brain (Magidenko 2008). We investigated the protective effects of Lcarnitine in cases of hippocampal damage due to experimental FA toxicitation in rats using physiologic, histopathologic and immunohistochemical techniques.

Materials and methods Twenty-four 300-320 g, 7-9-month-old Wistar albino male rats were used for our study. The research project and animal housing conditions were approved by the Ethical Committee for Animal Studies (approval 2010-3-11). Rats were obtained from the Fırat University Laboratory Animal Breeding Unit. The rats were assigned randomly to four groups. The first group was the control group (C); these animals were injected intraperiteonally (IP) once daily for 14 days with 1 ml sterile phosphate buffered saline (PBS). We injected the second group of animals IP with 10 mg/kg 37% FA (Merck, Darmstadt, Germany) once daily for 14 days. We injected the third group of animals IP with FA  0.5 g/kg L-carnitine (FA  0.5 LC) (Carnitene® Santa Farma Ilac A.S., I˙stanbul, Turkey) once daily for 14 days. We injected the fourth group of animals IP with FA  1 g/kg L-carnitine (FA  1 LC) once daily for 14 days. FA was diluted with 10% PBS in all cases. FA and L-carnitine doses were administered to the animals according to previous studies (Binienda and Ali 2001, Pitten et al. 2000). During the experimental period, the animals were fed laboratory rodent chow that contained 4.3% fiber, 3.8% fat, 15.5% crude protein and 2750 kcal/ kg metabolizable energy (Fay Feeds, Hatay, Turkey) and tap water ad libitum. At the end of the 14-day experimental period, animals were sacrificed under anesthesia using an intramuscular injection of ketamine (50 mg/kg) and xylazine (20 mg/kg). Animals were decapitated and samples of hippocampus were excised for measurement of MDA, GSH and SOD activities and for histopathological examination. For biochemical analyses, hippocampus tissue samples were placed in liquid nitrogen and stored at 80° C. The samples (200 mg tissue sample in

800 ml isotonic sodium chloride solution) were homogenized at 9000 rpm in an ultrasonic homogenizer (IKA). This homogenate then was centrifuged at 4025 x g) for 10 min (Sigma18-K) to obtain supernatants. MDA, GSH and SOD activities were determined using the supernatants by ELISA (ELX800 Biotech, Winooski, VT) and commercial kits (OxisResearch™, Foster City, CA) according to the manufacturers’ instructions.Some tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin using routine methods. Sections were cut 5–6 μm and stained with hematoxylin and eosin (H & E) (Luna 1968). The remaining tissues were de-waxed and rehydrated using routine methods for immunohistochemical staining. Immunostaining was carried out at room temperature using the avidin-biotin-peroxidase (ABC-P) method. Antigen retrieval was heated in citrate buffer, pH 6.0, for 10 min in microwave oven at 800 W. Endogenous peroxidase activity in tissue sections was blocked by applying 0.3% hydrogen peroxide in 0.01 M PBS containing 10% methanol to block nonspecific binding, and incubated with 5% normal goat serum prior to exposure to primary antisera. Sections were incubated with mouse anti- Cu/Zn-SOD (Novocastra Laboratories, Newcastle,UK) diluted 1:100 with PBS and with anti-ubiquitin (Sigma-Aldrich) diluted 1:200 with PBS overnight at room temperature. The sections were incubated with the rabbit anti-mouse biotinylated secondary antibody. Color labeling was developed by a final incubation step using 3-amino9-ethyl-carbazole (AEC, Dako, Glostrup, Denmark). Finally, sections were counterstained with Mayer’s hematoxylin, rinsed with tap water, and mounted with an aqueous mounting medium. For controls, the primary antibody was omitted and replaced by PBS. Following each incubation step, sections were washed thoroughly with PBS with the exception of the step after incubation with normal goat serum. All sections were examined by light microscopy (Olympus CX31).

Statistical analysis Differences among the groups were evaluated using the Duncan test using SPSS 17.0. Values for p  0.05 were considered significant.

Results The MDA level increased significantly only in the group treated with FA. The GSH levels were somewhat increased in the experimental groups, but the differences were not significant. The SOD level L-carnitine protection of hippocampus 337

Table 1. Plasma SOD, MDA and GSH levels in hippocampus tissue Groups C FA  0.5 g/kg L-carnitine FA  1 g/kg L-carnitine FA P

GSH (mM)

MDA (mM)

SOD (U/ml)

91.22  5.01a 76.51  5.54ab 79.53  5.32ab 66.37  6.01b 0.029

1.31  0.20b 2.12  0.18b 1.67  0.26b 4.10  1.06a 0.002

72.43  2.37 65.06  2.03 70.76  3.48 67.12  1.51 0.291

a,bMeans in the same column with different superscripts differ significantly (p  0.05). n  6; mean  error.

tended to decrease in all three experimental groups compared to the control. MDA, GSH and SOD levels in hippocampal tissue samples are summarized in Table 1. The histological appearance of hippocampal tissue of animals in the control group was normal. Anti-ubiquitin and anti-Cu/Zn-SOD expressions were not determined in the hippocampal tissues of the control group. In the FA group, cytoplasmic shrinkage of neurons and dark basophilic staining pyknotic nuclei (Fig. 1) were observed. In addition, near some of the degenerated neurons, microglial cell recruitment (satellitosis) and neuronophagia were observed. Degenerated neurons were stained by antiubiquitin (Fig. 2) and both the neurons and granular cells showed positive reactions with Cu /Zn-SOD antibody (Fig. 3). Any differences were observed in hematoxylin and eosin staining or immunoperoxidase staining between the LC  0.5 FA and FA  1 LC groups. Neuronal degenerative changes were minimal, and

cytoplasmic and nuclear staining was similar to the control group (Fig. 4). The neurons were not stained with anti-ubiquitin (Fig. 5) and only slightly stained with Cu/Zn-SOD antibody (Fig. 6).

Fig. 1. FA group showing shrunken neuronal cytoplasms with dark basophilic staining and nuclei (arrows). H & E.  40.

Fig. 2. FA group showing immunopositive staining of degenerated neurons (arrows) by anti-ubiquitin antibody. ABC-P.  40.


Discussion The hippocampus plays an important role in learning and memory, storing new information and tranferring memories from short-term to long-term; damage to the hippocampus may affect these functions adversely. FA from the environment is metabolized to formic acid by FA dehydrogenase (FDH) in the liver and red blood cells; FDH requires glutathione as a co-factor for this reaction. Therefore, reduction of the glutathione anti-oxidant increases the toxicity of FA (Smith 1992, Usanmaz et al, 2002). FA has carcinogenic effects on skin, eyes, central nervous system, gastrointestinal tract, testis, and menstrual functions (Zararsız et al 2004). Teng et al (2001) reported that low levels of FA caused oxidative stress in isolated

Biotechnic & Histochemistry 2014, 89(5): 336–341

Fig. 3. FA group showing immunopositive staining of degenerated neurons and granular cells (arrows) by antiCu/Zn-SOD antibody. ABC-P.  40.

Fig. 5. L-carnitine group showing staining by anti-ubiquitin antibody. ABC-P.  40.

rat hepatocytes. Many studies using animals indicate that exposure to FA inhibits learning, weakens memory and suppresses certain behaviors, but the mechanisms for these effects are not clear (Lu et al. 2008, Perna et al. 2001, Wang et al. 2008). FA causes oxidative stress in the brain (Matsuoka et al. 2010, Kus et al. 2004). We observed degenerative changes in hippocampal neurons caused by FA toxicity; similar results have been reported earlier (Dou et al. 2012, Gurel et al. 2005, Kus et al. 2004). We also observed satellitosis and neuronophagia. To determine the cellular damage in the hippocampus caused by FA and the potentially protective effect

of L-carnitine, we measured the level of the lipid oxidation product, MDA, which was significantly increased in the FA and C groups compared to the other two experimental groups. The increased MDA levels may be an indicator of oxidative damage to the hippocampus due to FA exposure. Lu et al. (2008) reported that learning and memory of rats deteriorated after FA inhalation. In our study, FA caused severe oxidative stress in brain neurons. Although FA administration produced a GSH level close to that of the control group, GSH level in the remaining two trial groups were somewhat increased compared to the control group, but the differences were not significant.

Fig. 4. L-carnitine group showing reduced degenerative changes in neurons and normal histological appearance (arrows). H & E.  40.

Fig. 6. L-carnitine group showing pyramidal cells stained by anti-Cu/Zn-SOD antibody (arrows). ABC-P.  40.

L-carnitine protection of hippocampus 339

We conclude that the decreased OD levels in the L-carnitine supplemented group indicate that L-carnitine suppressed oxidative stress in the brains of rats caused by FA. In support of our findings, the protective effects of L-carnitine on the brain and peripheral nervous system have been demonstrated both in vivo and in vitro (Manfridi et al. 1992, Mannelli et al. 2007). Tu’nez et al. (2007) reported that application of L-carnitine protected the brain against oxidative stress caused by thioacetamide intoxication in rats. Our histopathological and immunohistochemical findings demonstrated revealed that the addition of L-carnitine to the diet minimized the degenerative changes in neurons caused by FA toxicity. Earlier reports showed that neuronal degeneration and death due to 3-nitropropionic acid (Binienda and Ali 2001) and lead (Özsoy et al. 2011) were decreased by administering L-carnitine to rats. The Cu /Zn-SOD antibody is used to demonstrate oxidative damage to hippocampal neurons; granular cells showed intense staining in the FA group, but very light staining and few positive areas in the L carnitine groups. Like previous studies (Capucchio et al. 2010, Hazıroglu et al. 2010, Lowe et al. 1993, Özsoy and Hazıroglu 2010, Özsoy et al. 2011, Schweitzer et al. 1993), degenerated neurons were stained strongly in the FA group, but the FA  0.5 LC and FA  1 LC groups were not stained by this antibody and appeared similar to the control group. Ubiquitin is a protein that targets proteins for non-lysozomal degradation (Lowe et al. 1993, 1997) and during neuron destruction the antibody staining is intense (Klimaschewski 2003, Hazıroglu et al. 2010, Özsoy and Hazıroglu 2010, Özsoy et al. 2011). In our study, the degenerated neurons of the FA group stained strongly with anti-ubiquitin antibody; with L-carnitine addition, no staining of neurons was observed. We found that L-carnitine was an useful additive for preventing oxidative damage in hippocampus tissue due to FA intoxication and that it plays an important protective role for neurons.

Acknowledgment This research was supported by project Mustafa Kemal University Scientific Research Projects (1005 M 0112). Conflict of interest statement: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper 340

References Arıncı K, Elhan A (2001) Anatomi. 3rd ed., Günes Kitabevi, Ankara. pp. 318–321. Binienda ZK, Ali SF (2001) Neuroprotective role of L-carnitine in the 3-nitropropionic acid induced neurotoxicity. Toxico. Lett. 125: 67–73. Capucchio MT, Márquez M, Pregel P, Foradada L, Bravo M, Mattutino G, Torre C, Schiffer D, Catalano D, Valenza F, Guarda F, Pumarola M (2010) Parenchymal and vascular lesions in ageing equine brains: histological and immunohistochemical studies J. Comp. Pathol. 142: 61–73. Carrol MC, Core E (2001) Carnitine: A review. Comp. Cont. Educ. Pract. Vet. 23: 45–52. Cos˛kun T (1999) Karnitinin klinik önemi ve uygulamaları. In: Özalp I˙, Yurdakök M, Cos˛kun T, Eds., Pediatride Gelis˛meler, Sinem Ofset, Ankara. pp. 477–483. Cos˛kun T (2003) Karnitin. Pediatri Dergisi 25: 511–521. Dou XJ, Zhang Y, Wu YH (2012) Study on neurotoxicity of formaldehyde in mice. Toxicol. Environ. Health Sci. 4: 115–120. Frølich KW, Andersen LM, Knutsen A, Flood PR (1984) Phenoxyethanol as a nontoxic substitute for formaldehyde in long-term preservation of human anatomical specimens for dissection and demonstration purposes. Anat . Rec. 208: 271–278. Gurel A, Coskun O, Armutcu F, Kanter M, Ozen OA (2005) Vitamin E against oxidative damage caused by formaldehyde in frontal cortex and hippocampus: biochemical and histological studies. J. Chem. Neuroanat. 29:173–178. Hazıroglu R, Guvenc T, Tunca R, Ozsoy SY, Ozyildiz Z (2010) Evaluation of the age related changes in dog brains. Rev. Med. Vet. 161: 72–78. Imbus HR (1985) Clinical evaluations of patients with complaints related to formaldehyde exposure. J. Aller. Clin. Immunol. 76: 831–840. Klimaschewski L (2003) Ubiquitin-dependent proteolysis in neurons. News Physiol. Sci. 18: 29–33. Kus˛ I˙, Zararsız I˙, Yılmaz HR, Özdem Türkog˘lu A, Pekmez H, Sarsılmaz M (2004) the protective effects of melatonin hormone against exposure of formaldehydeınduced oxidative damage in prefrontal cortex of rats. E.Ü. J. Health Sci. 13: 1–7. Kus˛ I˙, Zararsız I˙, Ögetürk M, Yılmaz HR (2007) Formaldehit nörotoksisitesine bag˘lı hipokampusta gelisen oksidatif hasar ve melatonin hormonunun koruyucu etkisi: deneysel bir çalıs˛ma. Fırat Tıp Derg. 12: 256–260. Lowe J, Mayer RJ, Landon M (1983) Ubiquitin in neurodegenerative diseases. Brain Pathol. 3: 55–65. Lowe J, Lennoxm G, Leigh NP (1997) Disorders of movement and system degenerations. In: Graham DI, Lantos PL, Eds. Greenfield’s Neuropathology, 6th ed. Arnold, London. pp. 339–341. Lu Z, Li CM, Qiao Y, Yan Y, Yang X (2008) Effect of inhaled formaldehyde on learning and memory of mice. Indoor Air. 18: 77–83. Luna LG (1968) Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw-Hill Book Co., NewYork. p. 32.

Biotechnic & Histochemistry 2014, 89(5): 336–341

Manfridi A, Forloni GL, Arrigoni-Martelli, E, Mancia M (1992) Culture of dorsal root ganglion neurons from aged rats: effects of acetyl-L-carnitine and NGF. Int. J. Dev. Neurosci. 10: 321–329. Mannelli LDC, Ghelardini C, Calvani M, Nicolai R, Mosconi L, Vivoli E, Pacini A, Bartolini A (2007) Protective effect of acetyl-l-carnitine on the apoptotic pathway of peripheral neuropathy. Eur. J. Neurosci. 26: 820–827. Matsuoka T, Takaki A, Ohtaki H, Shioda S (2010) Early changes to oxidative stress levels following exposure to formaldehyde in ICR mice. J. Toxicol. Sci. 35: 721–730. Özsoy SY, Hazırog˘lu R (2010) Age-related changes in cat brains as demonstrated by histological and immunohistochemical techniques. Rev. Med. Vet. 161: 540–548. Özsoy SY, Özsoy B, Ozyildiz Z, Aytekin I (2011) Protective effect of L-carnitine on experimental lead toxicity in rats: a clinical, histopathological and immunohistochemical study. Biotech. & Histochem. 86: 436–443. Perkins JL, Kimbrough JD (1985) Formaldehyde exposure in a gross anatomy laboratory. J. Occup. Med. 27: 813–815. Perna RB, Bordini EJ, Deinzer-Lifrak M (2001) A case of claimed persistent neuropsychological sequelae of chronic formaldehyde exposure clinical, psychometric, and functional findings. Arch. Clin. Neuropsych. 16: 33–44. Pitten FA, Kramer A, Herrmann K, Bremer J, Koch S (2000) Formaldehyde neurotoxicity in animal experiments. Pathol. Res. Pract. 196: 193–198. Smith AE (1992) Formaldehyde. Occup. Med. 42: 83–88. Songur A, Özen OA, Sarsılmaz M (2001) Hipocampus. J. Med. Sci. 21: 427–431.

Stroup NE, Blair A, Erikso GE (1986) Brain cancer and other causes of death in anatomists. JNCI 6: 1217–1224. Schweitzer JB, Park MR, Einhaus SL, Robertson JT (1993) Ubiquitin marks the reactive swellings of diffuse axonal injury. Acta Neuropathol. 85: 503–507. Teng S, Beard K, Pourahmad J, Moridani M, Easson E, Poon R (2001) The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes. Chem. Biol. Interact. 130: 285–296. Tu´ nez I, Mun˜ oz MC, Medina FJ, Salcedo M, Feijo´ M, Montilla P (2007) Comparison of melatonin, vitamin E and L-carnitine in the treatment of neuro- and hepatotoxicity induced by thioacetamide. Cell Biochem. Funct. 25: 119–127. Usanmaz SE, Akarsu ES, Vural N (2002) Neurotoxic effects of acute and subacute formaldehyde exposures in mice. Env. Toxicol. Pharmacol. 11: 93–100. Walter P (2000) L-carnitine a vitamin-like substance for functional food. Ann. Nutr. Metab. 44: 75–96. Wang X L, Yuan F S, Zhang Z H (2008) Effect of formaldehyde inhalation on mice’s learning and memory. J. Env. Health 25: 400–402. Zararsız I˙, Kus˛ I˙, Çolakog˘lu N, Pekmez H, Yılmaz HR, Sarsılmaz M (2004) Formaldehit maruziyeti sonucu sıçan akcig˘erinde olus˛an oksidatif hasara kars˛ı melatonin hormonunun koruyucu etkisi: is˛ık mikroskobik ve biyokimyasal çalıs˛ma. Van Tıp Derg. 11: 105–112. Zeyner A, Harmeyer J (1999) Metabolic functions of L-carnitine and its effects as feed additive in horses. A review. Arch. Anim. Nutr. 52: 115–138.

L-carnitine protection of hippocampus 341

Copyright of Biotechnic & Histochemistry is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.

The protective effect of L-carnitine against hippocampal damage due to experimental formaldehyde intoxication in rats.

We investigated the protective effects of L-carnitine on hippocampus tissue damage in rats during experimental formaldehyde (FA) intoxication. Male Wi...
778KB Sizes 0 Downloads 0 Views