Trihexyphenidyl (THP; Benzhexol) is a synthetic anticholinergic antiparkinson drug that is used to ameliorate extrapyramidal symptoms caused by either Parkinson’s disease or antipsychotic drugs . Moreover, it has excellent anticonvulsant properties by exerting both cholinergic and glutamatergic antagonism . It also demonstrated improvement of dystonia and sialorrhea in children with cerebral palsy .
Recent study proved that the occurrence of THP (7–28 mg/day) abuse is linked to its hallucinogenic and euphoric effects in young males with deviant behavior . THP abusers are characterized by being young, unmarried, smokers, with poor employment, educational, and social skills. In addition, they often have past and concurrent history of multiple drug abuse and genetic loading of mental disorders [5,6]. Drug abuse provides the homeless children with some relief from the pressures of the street, and enables them to endure pain, violence, and hunger. The abusers usually prefer anticholinergics over other drugs because they are readily available and cheap . THP abuse has been also observed among patients with schizophrenia helping them to feel and function well, relieve negative symptoms (hostile-suspiciousness), and/or alternatively for its nonspecific stimulant effects . Evidence of THP abuse by patients with chronic schizophrenia up to 200 mg/day, to achieve an euphoric effect, has been reported .
The medicinal misuse of THP has been observed among several segments of society in Sydney, Australia , Jordan , and Brazil . Surprisingly, THP has been recognized as the third commonest drug abused among students of some secondary schools in Assiut, a city in south Egypt . Recently, THP abusers have been diagnosed accidentally among the comatosed patients admitted in Mansoura Emergency Hospital in Egypt.
THP exerts a sustained improvement of motor function in hemiparkinsonian rats chronically treated with low doses of THP  and in human . However, the effect of THP abuse on the motor area of the frontal cortex, which is responsible for the voluntary control of muscle movements, has not been adequately investigated. Therefore, the present study was designed to identify the structural alterations of the motor area, induced by different single doses of TPH in rats.
Materials and methods
THP (Benzhexol) was obtained from the Nile Co. for Pharm. Cairo-A. R. E. (Egypt) as Parkinol tablets (5 mg).
The current study was carried out on 48 adult male albino rats weighing 180–220 g. The animals were obtained from the Urology and Nephrology Center, Mansoura University, Egypt. They were kept in adequate ventilation and temperature with regular 12 h light/ 12 h dark cycle in plastic cages and were allowed a free access to standard laboratory food and water ad libitum.
The rats were divided into four main groups (12 rats each):
- Group I: Control rats that received intraperitoneal injection of 0.5 ml saline.
- Group II: The rats received THP in a dose of 0.2 mg/kg body weight (equivalent to the human therapeutic dose) .
- Group III: The rats received THP in a dose of 0.5 mg/kg body weight .
- Group IV: The rats received THP in a dose of 1.0 mg/kg body weight .
The THP doses in groups III and IV represent two different doses of drug abuse . The THP drug was dissolved in sterile saline solution and administered as a single dose by intraperitoneal injection.
Measurement of serum trihexyphenidyl level
After 6 h of injection, blood samples were collected from the retro-orbital sinus of all groups. The serum was separated by centrifugation and frozen at −20°C for subsequent biochemical analysis. Serum THP level was determined using radioimmunoassay. Quantification was achieved at 254 nm over concentration ranges of 1.0, 2.0, 4.0, 6.0, and 8.0 μg/ml .
All animals were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally), then rats were perfused through the left ventricle, first with 500 ml of 0.1 mol/l phosphate buffer (pH 7.4), then with phosphate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde. The left motor area (in the precentral gyrus of the frontal cortex) was dissected rapidly, kept in 10% neutral-buffered formalin and processed for light microscopic (LM) study. Paraffin sections (5 μm thick) were prepared and stained with H&E staining. For transmission electron microscopic study, small pieces (1 mm3) from the motor area were rapidly cut and immediately fixed in 0.1 mol/l phosphate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde at 4°C overnight in phosphate buffer in the refrigerator. Specimens were then postfixed in 1% osmium tetroxide for 1h at 4°C. After repeated washing, specimens were dehydrated in graded ethanol and embedded in Epon. Semithin sections (1 μm) were cut by ultramicrotome (Leica ultracut UCT, Germany) using glass knife, stained with 1% toluidine blue and examined with light microscope to select areas for subsequent ultramicrotomy. Ultrathin sections (60–80 nm) were cut using diamond knife and double stained with uranyl acetate and lead citrate . Sections were examined and photographed with transmission electron microscope (JEOL 100 CX, Japan), Faculty of Science, Alexandria University.
Using H&E-stained sections of the left frontal cortex, 10 fields from each group were randomly chosen and examined by light microscope using a magnification of × 400 to count the number of apoptotic pyramidal cells based on the morphological changes in the cells, such as cell shrinkage, acidophilic cytoplasm, and chromatin condensation [18,19].
The electron microscopic images were digitized using HP scanner (USA) at 600 dpi optical resolution and saved in BMP format for image analysis.
Image analysis was performed on Intel Core i3-based computer with windows Xp SP3 and Video Test Morphology software (Russia) with a built-in routine for morphological and optical parameters analysis.
Measurement of the mean mitochondrial intensity
To assess the degree of mitochondria damage, the mitochondria were manually extracted from the source image and automatically analyzed for mean mitochondrial intensity.
Measurement of the index of circularity of the myelinated axons
To assess the degree of axons irregularity, the axons were manually extracted and analyzed for determining circularity index. The index of circularity (IC) was calculated as the ratio between the measured myelinated nerve fiber area (A) and the area of a circle with the same circumference (B). This feature was calculated with equation IC=A/B .
The data were analyzed using Student’s t-test and paired t-test by a computer statistical package (SPSS program version 16, Germany). Results were presented as mean ± SD. A P-value of less than 0.05 was considered significant.
Group I (control group)
In H&E-stained sections, the frontal cortex motor area of group I was covered by pia matter containing blood vessels. Six layers were identified in the cerebral cortex; outer molecular layer, external granular layer, external pyramidal layer, inner granular layer, inner pyramidal, and the polymorphic layer (Fig. 1). The pyramidal cells of the inner pyramidal layer showed vesicular nuclei, basophilic cytoplasm, and long apical dendrites, whereas the granular cells, perineural neuroglia cells, and blood capillaries were scattered among neurons. The granular cells showed large open face nuclei with prominent nucleolus and little cytoplasm. The pink-stained background, the neuropil, appeared as a mat of neuronal and glial cell processes (Fig. 2). Electron microscopic study of rat motor area revealed the large pyramidal cells with euchromatic rounded nuclei and long apical dendrites (Fig. 3). Their cytoplasm contained numerous free ribosomes among the rough endoplasmic reticulum (rER) cisternae (Nissl granules), mitochondria, lysosomes, and Golgi complex (Fig. 4). The myelinated axons (axons) in the neuropil showed regular arrangement of the myelin lamellae, and the axoplasm contained mitochondria and neurofibrils (microtubules and microfilaments) (Fig. 5). A perineural neuroglia cells were also seen (Fig. 3).
Apart from some dilated Golgi saccules (Fig. 6), light and electron microscopic examination revealed that the frontal cortex motor area of group II rat was nearly similar in structure to that of the control group.
LM examination of rat frontal cortex motor area of group III showed some shrunken pyramidal and granular cells with pyknotic nuclei and acidophilic cytoplasm, most probably apoptotic cells. Most of the pyramidal cells and granular cells appeared normal (Fig. 7). Electron microscopic examination showed that some pyramidal cells exhibited irregular heterochromatic nuclei with multiple heterochromatic clumps, dilated Golgi saccules, apparent decrease in free ribosomes, dilated rER cisternae, multiple lysosomes and cristolysis of the mitochondria which present in both the cytoplasm and the long apical process (Figs 8 and 9). Apoptotic pyramidal cells were seen with multiple electron-dense bodies, most probably fragmented nuclei (karyorrhexis) in their cytoplasm. In addition, the apoptotic cells showed dilated Golgi saccules, cristolysis of the mitochondria and numerous free ribosomes among the rER cisternae (Fig. 10). The axons exhibited irregular contour and multiple splitting of their myelin lamellae with wide spaces in between (Figs 11 and 12). Axoplasmic vacuoles were also observed (Fig. 11).
LM examination of rat motor area of group IV (Figs 13 and 14) showed an apparent increase in the number of the apoptotic pyramidal and granular cells compared with group III (Fig. 7). The apoptotic cells were shrunken and surrounded by halos and exhibited condensed darkly stained nuclei, acidophilic cytoplasm (Figs 13 and 14). Focal acidophilic necrotic areas with microglial infiltration were also demonstrated (Fig. 14). Electron microscopic examination showed pyramidal cells with dilated Golgi saccules, apparent decrease in the free ribosomes, dilated rER, cristolysis of the mitochondria, and irregular hyperchromatic nuclei (Fig. 15). Other pyramidal cells, most properly apoptotic cells, appeared shrunken with vacuolated cytoplasm, degenerated cell organelles, degenerated mitochondria, blebbing of the cell membrane, and were surrounded by halos (Figs 16 and 17). In addition, the nucleus appeared irregular, hyperchromatic with prominent nucleolus, and evident invagination of the nuclear membrane (Figs 16 and 17). Furthermore, some pyramidal cells appeared crumpled and degenerated (Fig. 18). The axons showed degenerative changes such as irregular contour, multiple focal loss of the myelin lamellae (Figs 18 and 19), axoplasmic vacuoles, and abnormal electron-dense mitochondria (Fig. 19). Extravasation of red blood corpuscles were also seen in the frontal cortex (Fig. 18).
The current study revealed a nonsignificant difference between groups I and II regarding the number of apoptotic pyramidal cells, mitochondrial intensity, and the IC of the myelinated axons (Table 1). Groups III and IV revealed a significant increase (P < 0.01) in the mean serum level of THP, the number of apoptotic pyramidal cells as well as mitochondrial intensity and a significant decrease (P < 0.01) in the IC of the myelinated axons (Table 1).
The therapeutic dose of TPH, 8–20 mg/day, is more effective than other drugs in improving motor functions in Parkinson’s disease . It has been recorded that the use of THP improves one or more motor areas, mainly arm, hand, and oromotor function in children with dystonic cerebral palsy . Several studies have described the neuropsychiatric, cognitive adverse events, hallucinations, confusion, memory problems, restlessness, and drug abuse which frequently occur with THP therapy and are the main reason for the drug withdrawal than lack of efficacy [1,4,6,9,10,21]. In addition, THP drug showed dose-dependent amnesic effects in the parkinsonian and schizophrenic patients . In contrast, the effect of THP abuse on the motor area of the frontal cortex has not been adequately investigated. Therefore, the present study was designed to identify the structural alterations of the frontal cortex motor area, induced by different single doses of TPH in rats.
In group II, administration of 0.2 mg/kg of TPH (equivalent to the human therapeutic dose) resulted in a nonsignificant difference, in comparison to the control group, regarding the number of apoptotic pyramidal cells, intensity of mitochondria, and the IC of the myelinated axons. This result could be explained on the basis that the low dose of THP exhibits an antioxidant activity which significantly reduces the intracellular reactive oxygen species formation, protects against oxidative cell damage, and the mitochondria membrane potential changes recognized as an early event in lethal cell insult (e.g. apoptosis) . An in-vitro study in rat neuroendocrine cells demonstrated that THP significantly attenuated the morphological alterations such as cell shrinkage, mitochondrial changes, and membrane blebbing, the typical characteristics of apoptotic cell death .
In groups III and IV, administration of 0.5 and 1.0 mg/kg TPH, respectively (representing two different high doses of TPH drug abuse) induced a significant increase (P < 0.01) in the mean number of apoptotic pyramidal cells in comparison to groups I and II. In addition, microscopic examination depicted apoptotic changes in the pyramidal cells. Similar apoptotic changes such as cellular shrinkage, typical nuclear condensation, fragmentation in the pyramidal neurons and cytoplasmic blebbing have been reported in rhesus monkey  and rat [26,27] brain as a result of drug and septic neurotoxicity, respectively. It was demonstrated that the early phase of apoptosis is characterized by invagination of the nuclear membrane, whereas the late phase shows condensed chromatin segregated along the margin of the nuclear membrane, densely packed organelles, including mitochondria and electron-dense vesicles in the cytoplasm [19,28].
Cristolysis of the mitochondria was demonstrated in both groups III and IV, associated with a significant increase (P < 0.01) in the mean mitochondrial intensity. It has been documented that mitochondrial damage (mitochondria with broken cristae) is a contributory factor to several physiological processes of aging and pathological progression in the central nervous system (CNS), leading usually to some neurodegenerative disorders such as Alzheimer’s diseases [29,30]. A subsequent decrease in mitochondria membrane potential may release an apoptosis inducing factor which activates caspase protease(s), causes nuclear condensation and cytoplasmic fragmentation .
Ultrastructural study of axons revealed irregular contour, multiple splitting of the myelin lamellae in group III. In addition, group IV showed degenerative changes in the form of axoplasmic vacuoles, abnormal electron-dense mitochondria, and multiple focal demyelination of the axons. Furthermore there was a significant decrease (P < 0.01) in the mean IC. Several mechanisms have been suggested to explain myelin alterations in the rat brain, such as decreased myelin-associated glycoprotein, autoantibodies to myelin basic protein, and myelin damage induced by nitric oxide .
In group IV, LM examination of the motor area revealed focal acidophilic necrotic areas with microglial infiltration. Electron microscopic examination revealed degenerated crumpled pyramidal cells and extravasation of red blood corpuscles. These structural changes could explain the underlying brain lesion of the comatosed abusers admitted to Mansoura Emergency Hospital. In line with the present results, previous clinical research reported that the motor movements of children with hyperkinetic dystonia (excess involuntary movements) were worsened after a high-dose of THP (0.75 mg/kg/day) therapy . It was proved that the spectrum of anticholinergic CNS adverse effects ranges from drowsiness to hallucinations, severe cognitive impairment, and even coma . Significant memory impairment after a single (1.0 mg) dose of THP has been reported . However, a human study reported a case of successful monotherapy of rubral tremors by a single high daily dose of THP (38 mg) without severe or uncomfortable side effects .
Several mechanisms were suggested for THP toxicity. It was hypothesized that THP inhibits the cortical cholinergic system and significantly impairs endothelium-dependent relaxations of cerebral arteries, decreases regional cerebral blood flow and oxygen metabolic rate in the cerebral cortex . In addition, anticholinergic agents may penetrate the blood–brain barrier resulting in CNS side effects .
In conclusion, in the light of the results of the present study, it has been found that TPH induces dose-dependant structural changes in rat motor area which could explain the brain pathologic process leading to abnormal brain function. Therefore, it is recommended that THP prescription should be under strict supervision.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2011 The Egyptian Journal of Histology
anticholinergic agents; drug abuse; frontal cortex; motor area; rats; trihexyphenidyl