1. Baclofen systemic toxicity: Experimental histopathological and biochemical study  

2. Sahar Y. Issa, MD. , Essam M. Hafez, MD. Asmaa S. El-Banna. , Safaa M
Sahar Y. Issa, MD *, Essam M. Hafez, MD ** Asmaa S. El-Banna*, Safaa M. Abdel Rahman ***, Maha K AlMazroua****, Mostafa Abo El-Hamd**
* Department of Forensic Medicine &Clinical Toxicology, Faculty of Medicine, Alexandria University
**Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Minia University ***Central Laboratory, Alexandria **** Dammam Poison control center, MOH-KSA.

4. Introduction
Baclofen (parachlorophenyl gamma-aminobutyric acid) is a potent agonist of the neurotransmitter gamma-aminobutyric acid (GABA).
The centrally acting effect of the drug has been used mainly to treat spasticity of spinal origin, in addition to the treatment of alcohol dependence.
More recently, off-label use of high-dose baclofen markedly increased. It has been lately used to treat chronic hiccups of different etiologies, as well as anxiety disorders.

5. Introduction
Baclofen is well absorbed in the gastrointestinal tract, producing the determined therapeutic levels of 80–400 ng/ml.
The main method of drug excretion is through the kidney (70–80%), whereas the rest is metabolized in the liver or processed through the gastrointestinal tract.
Minute fraction of the drug only is capable of crossing the blood brain barrier, leading to the desired effects.

6. Introduction
The drug's half-life is nearly 6.8 hours. Patients with known impairment of renal functions have longer half-life with more crossing of the blood brain barrier.
The generalized central nervous system (CNS) depression effects will lead to fatigue, syncope, hypotension, ataxia, psychological disturbances, and cardiovascular and respiratory depression.

7. In patients with inadequate renal function, reports of toxic side effects with the initial dosing of 5mg three times a day within only a few days has been reported.
Baclofen toxicity usually improve with hemodialysis with excretion rates similar to normal renal function.
Every drug has been associated with hepatotoxicity most likely due to the fundamental role of the liver in drug metabolism. Drug metabolites excreted from kidneys may also cause cellular damage ending in renal dysfunction

9. OBJECTIVES biochemical changes and histopathological changes in rats.
The aim of the present study was to Investigate the toxicity of Baclofen as manifested by:
biochemical changes and
histopathological changes in rats.

10. Methods
I- Animals
Thirty healthy adult male albino rats weighting about grams were obtained from the animal house in faculty of Agriculture, Alexandria University.
All animals were allowed free access to distilled water and laboratory chow ad libitum.
To avoid stress of isolation or overcrowdings, 5 rats were housed per cage.

11. Methods
They were left freely moving in their cages for two weeks with 12 hour dark to light cycle for acclimatization before starting the experiment.
Experimental procedures were performed in accordance with the guide of the care and use of laboratory animals approved by the Ethics committee of Alexandria University, the fewest number of animals estimated to obtain valid results were used and painful procedures were conducted with appropriate sedation to avoid pain and stress.

12. Methods Baclofen, was purchased from Sigma Chemical Company.
II-Drug:
Baclofen, was purchased from Sigma Chemical Company.
All doses of Baclofen were delivered to rats by using the gavage technique.
At the end of each experimental time and using ether anesthesia, all animals were sacrificed after 24 hours of the last dose.

13. Methods III. Experimental design:
Group I: (negative control): (5 rats) were kept in a quite group non-stressful environment, provided with food ad libitum and free access to water for one month.
Group II: (positive control): (5 rats) each animal received 1ml/day normal saline 0.9% orally. They were kept throughout the experiment under the same conditions for one month.

14. Methods
Group III (Baclofen acute toxicity group 20 rats): Each animal received a single dose of LD50 of baclofen orally by gavage. It equals 145mg/kg b wt. The rats were observed for acute toxicity manifestations as well as for LD50 deaths.
Group IV (Baclofen Dependence group 20 rats): Each animal received baclofen (1/10th LD50) in gradually increasing doses for one month. Dependence was induced by giving the calculated dose of baclofen. The dose was then gradually increased by adding the initial calculated dose every three days till the end of the study month.

15. Methods IV-Biochemical assay:
3 ml of blood was collected from all animals. The collected samples were centrifuged for 10 minutes at 5000 rpm. Separated clear serum was collected to assay Liver function tests including AST, ALT, ALP and GGT.
RFTs namely; SC, BUN, and CK were checked.
PT, & PTT were measured in seconds.
Total protein (TP), Cardiac Troponin I (CTnl) and glucose level were measured in all studied groups.

16. Methods VI- Histopathological Studies:
At the end of the designed study scarification and decapitation of treated rats were done.
The right lobe of the liver and right kidney, lungs, stomach, and brain tissues of all rats were excised, fixed in buffered 10% formalin solution for 24 hours and embedded in paraffin wax, then sectioned and stained with Haematoxilin and Eosin stain (H&E) for histological examination under light microscope.

17. Statistical Analysis Biochemical Data were presented as the mean ± SD.
Statistical analysis was performed using SPSS V 22.
The 0.05 level of probability was used as the criterion of significance.

18. Results
Highly significant elevation (p < 0.0l) in the level of creatinine kinase (CK) was observed in both groups treated with Baclofen. Elevated level of blood urea nitrogen (BUN), serum creatinine (SC) were recorded in group III when compared to all other groups.
A significant difference in blood urea nitrogen (BUN), serum creatinine (SC), biochemistry parameters was detected between the control group and all other groups as given in table (1).

19. Table (1): Effect of Baclofen administration on biochemical laboratory findings in adult male rats
Test
Groups I & II
(Negative and positive Control Groups)
Group III
(Baclofen acute toxicity group)
Group IV
(Baclofen Dependence group)
Reference Interval
Total protein (TP) in g/dl
6.4±0.29
8.1±0.31
7.2±0.7
Glucose in mg/dl
72.5±4.1
76.3±4.1
89.4±6.2
70-105
Blood urea nitrogen (BUN) in mg/dl
10.3±1.3
21.9±1.8*
29.4±4.4**
12-26
Serum creatinine (SC) in mg/dl
1.3±0.1
2.1±0.4*
1.8±0.19
1.2-2
Creatinine kinase (CK) in U/L
94±18.7
452.3±12.1**
461.5±24.2**
0-350
Aspartate aminotransferase (AST) in U/L
13.61±3
52±7.4*
64.2±5.1**
0-35
Alanine aminotransferase (ALT) in U/L
9.20±3.4
86.9±15**
92.9±32.7**
7-56
Alkaline phosphatase (ALP) in U/L
32.2±9.1
68.7±13.9*
165.3±23.4**
30-120
Gamma-glutamyl transferase (GGT) in U/L
18.1±4.0
64.7±9.3*
68.1±8.2*
0-42
Cardiac troponin I (CTnl) in µg/L
5.1±1.1
12±2.7*
19±5.6*
0-10
Prothrombin time (PT) in seconds
8.7±1.2
15.2 ±1.1*
21.1 ±1.2**
8-10
Partial thromboplastin time (PTT) in seconds
31.6±3.4
41.2 ±4.7
67.62±2.4*
32-42

20. Results
The renal tissue histopathological examination revealed, eosinophilic intranuclear inclusions in tubular cells, found in the majority of proximal tubular cells.
Changes were not associated with degenerative or functional changes in group III as seen in (figure 1 -C). Group IV rats showed tubular degeneration with ongoing regenerative hyperplasia, showing epithelial multi-layering. (figure 1-D)

21. A B
Figure 1: Renal Tissue in different groups: (A) Grade 0.0: Normal architecture of the renal tissue in group I (negative control) H&E (X 200) - (B) Grade 0.0: Normal architecture of the renal tissue in group II (positive control) H&E (X 250) - (C) Grade 2A.3: Eosinophilic intranuclear inclusions in tubular cells, found in the majority of proximal tubular cells that are not associated with degenerative or functional changes in group III (Baclofen acute toxicity). H&E (X400 magnification to show eosinophilic inclusions) - (D) Grade 5.1: Tubular degeneration with ongoing regenerative hyperplasia in group IV (Baclofen dependence) showing hyperplastic tubules with epithelial multilayering H & E (X150).

22. Results
The hepatic tissue histopathological examination revealed, Coagulative necrosis of centrilobular hepatocytes with absence of inflammatory response in group III (Baclofen acute toxicity).
Centrilobular (periacinar) necrosis & Marked Perivenous area of coagulative necrosis and occasional cells with cytoplasmic vacuolation and ballooning, with minimal inflammatory reaction were noted in group IV (Baclofen dependence

23. Figure 2: Liver Tissue in different groups: (A) Grade 0
Figure 2: Liver Tissue in different groups: (A) Grade 0.0 Normal architecture in group I (negative control) H&E (X 200) - (B) Grade 0.0 Normal architecture in group II (positive control) H&E (X200) - (C) Grade 4.2: Coagulative necrosis of centrilobular hepatocytes with absence of inflammatory response in group III (Baclofen acute toxicity) H & E (X100) - (D) Grade 4.3: Centrilobular (periacinar) necrosis . Marked Perivenous area of coagulative necrosis and occasional cells with cytoplasmic vacuolation and ballooning, with minimal inflammatory reaction in group IV (Baclofen dependence H & E (X200)

24. Results
The Brain tissue histopathological examination revealed, Normal architecture of the brain tissue in the substantia nigra in group III with mild congestion (Baclofen acute toxicity).
Shrunken eosinophilic neurones with pyknotic nuclei were noted in the substantia nigra in group IV (Baclofen dependence).

25. Figure 3: Brain Tissue in different groups: (A) Grade 0
Figure 3: Brain Tissue in different groups: (A) Grade 0.0: Normal architecture of the brain tissue in the substantia nigra in group I (negative control) H&E (X 200) - (B) Grade 0.0: Normal architecture of the brain tissue in the substantia nigra in group II (positive control) H&E (X 200) - (C): Normal architecture of the brain tissue in the substantia nigra in group III with mild congestion Grade 1.2 (Baclofen acute toxicity) H&E (X 200) - (D) Grade 5.3: Shrunken eosinophilic neurones with pyknotic nuclei in the substantia nigra in group IV (Baclofen dependence) H&E (X100).

26. Results
The pulmonary tissue histopathological examination revealed, Pulmonary oedema, alveoli filled with weakly eosinophilic material and alveolar wall capillaries appear dilated with slight inflammatory reaction and little evidence of degenerative destructive changes in group III (Baclofen acute toxicity).
Congestion, inflammation, alveolar wall degeneration and focal fibroblast proliferation were noted in group IV (Baclofen dependence).

27. Figure 4: Lung Tissue in different groups: (A) Grade 0
Figure 4: Lung Tissue in different groups: (A) Grade 0.0 Normal architecture in group I (negative control) H&E (X 400) - (B) Grade 0.0 Normal architecture of the pulmonary tissue in group II (positive control) H&E (X400) - (C) Grade 4.3: Pulmonary oedema, alveoli are filled with weakly eosinophilic material and alveolar wall capillaries appear dilated with slight inflammatory reaction and little evidence of degenerative destructive changes in group III (Baclofen acute toxicity) H & E (X200 magnification) - (D) Grade 5.2: Congestion, inflammation, alveolar wall degeneration and focal fibroblast proliferation in group IV (Baclofen dependence). H & E (X200)

28. Results
The gastric tissue histopathological examination revealed, Normal architecture of the gastric tissue in group III (Baclofen acute toxicity).
Superficial mucosal necrosis in the glandular stomach of group IV rats (Baclofen dependence) , were noted in addition to oedematous sub mucosa.

29. Figure 5: (A) Grade 0.0: Normal architecture of the gastric tissue in group I (negative control) H&E (X200). - (B) Grade 0.0: Normal architecture of the gastric tissue in group II (positive control) H&E (X 250) - (C) Grade 0.0: Normal architecture of the gastric tissue in group III (Baclofen acute toxicity) H&E (X 250) - (D) Grade 5.2: Superficial mucosal necrosis in the glandular stomach of group IV rats (Baclofen dependence). The sub mucosa is oedematous. H & E (X200).

30. Conclusion
Baclofen toxicity is an under diagnosed toxicological emergency.
Physicians should consider baclofen toxicity in users having hepatorenal dysfunction, presenting with altered mental status, bradycardia and hypotension. To our knowledge this is the first research studying histopathological toxicity findings in experimental rats. Further investigations are recommended.