Endocytosis in chronically denervated mouse skeletal muscle. A biochemical and ultrastructural study with horseradish peroxidase.

Endocytosis of horseradish peroxidase was studiedin vivo in innervated mouse extensor digitorum longus and soleus muscles and in the same muscles 6 days after surgical denervation, by a combination of biochemical and morphological techniques. Biochemical determination of peroxidase activity in extensor digitorum longus and soleus muscles at different times after a single intravenous injection of horseradish peroxidase followed by extensive washing of the muscles, showed that peak levels of horseradish peroxidase in muscle tissue were obtained 2 h after injection. The uptake of horseradish peroxidase in denervated extensor digitorum longus and soleus muscles was approximately three times greater than in innervated control muscles. Light and electron microscopic examination of denervated and innervated extensor digitorum longus muscles, demonstrated that horseradish peroxidase was present in the extracellular space, i.e. between muscle fibers and in transverse tubules already 30 min after an intravenous horseradish peroxidase injection. Light-microscopic examination, 2–4 h after an intravenous injection of horseradish peroxidase, revealed that a population of denervated muscle fibers contained horseradish peroxidase in bodies or vacuoles restricted to segments of the fibers. Such bodies, containing horseradish peroxidase, were not observed in innervated muscle fibers. Ultrastructural examination showed that these horseradish peroxidase-bodies were limited by a single membrane and located adjacent to transverse tubules between myofibrils showing early signs of degeneration. Coated or uncoated vesicles of about 50–100 nm appeared to originate from horseradish peroxidase-labelled transverse tubules in denervated muscle fibers.These findings suggest that endocytosis (micropinocytosis) occurs from transverse tubules in segments of denervated muscle fibers where signs of degeneration can be observed. Following the entry of horseradish peroxidase, the tracer accumulates in heterophagosomes and secondary lysosomes of diverse appearance.     

Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents.

The use of alkaline phosphatase in an immunoenzymatic procedure for the localisation of antigens in paraffin sections or cell smears is described. The results of this method, when applied to the detection of immunoglobulins, lysozyme, or lactoferrin, were comparable in intensity and clarity to those obtained with the PAP immunoperoxidase procedure. Furthermore, double immunoenzymatic labelling (with alkaline phosphatase and peroxidase) of two cellular constituents in a tissue section is possible, the brown peroxidase reaction product contrasting well with the blue alkaline phosphatase product. Since the two antibody 'sandwiches' are applied simultaneously rather than sequentially the total duration of this double immunostaining procedure is only a few minutes longer than that required for detection of a single antigen. It was also found that the unlabelled antibody immunohistological procedure (whether used in conjunction with alkaline phosphatase or peroxidase) can be shortened without loss of sensitivity by carrying out two of the incubation steps simultaneously.     

Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies

Simultaneous detection of multiple tissue epitopes with an overlapping distribution pattern by monoclonal antibodies is sometimes needed for routine immunohistological evaluations. Therefore, multistep double and triple immunoenzymatic methods using antibodies from the same species or Ig (sub)class have been developed. Since only commercially available monoclonal antibodies (either unlabelled, biotinylated or as fluorescein conjugate) have been used, the techniques may be regarded as generally applicable. The staining protocol for double staining consists of six incubation steps: (1) unlabelled monoclonal antibody 1; (2) enzyme I-conjugated anti-mouse Ig; (3) normal mouse serum--for blocking; (4) fluoresceinated monoclonal antibody 2; (5) rabbit anti-fluorescein isothiocyanate--employing the fluorochrome as hapten; (6) enzyme II-conjugated anti-rabbit Ig. For enzymes I and II, peroxidase, alkaline phosphatase and beta-galactosidase can be applied; excellent results were obtained with the following colour combinations: peroxidase activity in red/alkaline phosphatase in blue and beta-galactosidase in green/alkaline phosphatase in violet. Moreover, this double staining method can be extended to provide an immunoenzyme triple staining technique by mixing biotinylated monoclonal antibody 3 and avidin-biotin enzyme III complex with the steps 4 and 5 reagents, respectively. In this way three tissue epitopes can simultaneously be detected clearly and selectively in green (beta-galactosidase), blue (alkaline phosphatase) and red (peroxidase).     

High stability of microRNAs in tissue samples of compromised quality

Degradation of tissue samples limits performing RNA-based molecular studies, but little is known about the potential usefulness of samples of compromised quality for studies focused on miRNAs. In this work we analyze a series of cryopreserved tissue samples (n?=?14), frozen samples that underwent a severe thawing process (n?=?10), and their paired formalin-fixed paraffin-embedded (FFPE) tissue samples (n?=?24) from patients with breast cancer obtained during primary surgical resection and collected in 2011. Quality and integrity analyses of the total and small fraction of RNA were carried out. Recovery of specific RNA molecules (miRNAs hsa-miR-21, hsa-miR-125b, and hsa-miR-191; snoRNA RNU6B; and mRNAs GAPDH and HPRT1) was also analyzed by quantitative RT-PCR. Our results suggest that visualisation of the small RNA electrophoretic profiles obtained using the Agilent 2100 bioanalyzer makes it possible to differentiate between the three groups of samples (optimally frozen, thawed, and FFPE). We demonstrate that specific miRNA molecules can be similarly recovered from different tissue sample sources, which supports their high degree of stability. We conclude that miRNAs are robustly detected irrespective of the quality of the tissue sample. In this regard, a word of caution should be raised before degraded samples are discarded: although prior quality assessment of the biological material to be analyzed is recommended, our work demonstrates that degraded tissue samples are also suitable for miRNA studies.     

Immunohistochemical detection of fibronectin using different fixatives in paraffin embedded sections

Different fixatives and immunohistochemical methods were tested for detection of fibronectin in various paraffin embedded tissues: rat kidney, spleen, gastro-intestinal tract, muscle, normal and fibrotic liver and human skin. Using cryostat sections, localisation with immunofluorescence and peroxidase technics comparable to those obtained in unfixed tissue sections, could be obtained with the following fixatives: 10% formalin in PBS containing 4% sucrose; 96% ethanol; 96% ethanol + 1% acetic acid; a series of ethanol solutions of increasing strength: 70-80-96%. These fixatives also proved to be the best for paraffin embedding. Without enzyme digestion, however, satisfactory results could not be obtained with either indirect peroxidase or immunofluorescence methods in paraffin embedded tissues. Following digestions with the enzymes at the concentrations described in the literature, the alteration of tissues made the morphological localization of fibronectin difficult. The self-sandwich peroxidase method following a gentle pepsin digestion gave results closest to those of unfixed cryostat sections; however a slight increase in background staining was observed but without interfering with the evaluation of results.     

Organotypic culture of embryonic electromotor system tissues from Torpedo marmorata

An explant culture system has been used to study the electric organ and electric lobe tissues of Torpedo marmorata at different stages during the development of the electromotor system.The myotubes in tissue expiants, taken from the electric organ primordia of 33–38 mm body-length embryos prior to electrocyte differentiation, contract spontaneously on explantation and have electrogenic membranes. The myotubes subsequently lose these properties in vitro and can differentiate in the absence of neural tissue into immature electrocytes which have morphologically characteristic postsynaptic membranes.Isolated expiants of differentiated electric organ tissue from 60–100 mm body-length embryos can be maintained for 3 to 4 weeks in vitro but cellular outgrowth is minimal. In contrast, a rapid, dense outgrowth of cells and a subsequent regeneration of myotubes occurs when differentiated electric organ explants are co-cultured with electric lobe tissue from embryos of the same stage. Cellular outgrowth from differentiated electric-organ tissue expiants can be stimulated by spinal cord, medulla, cerebellum and heart tissues but a subsequent regeneration of myotubes has not been observed. Myotube regeneration in the presence of electric lobe tissue is maximal with tissue from 60–80 mm body-length embryos. The myotubes that regenerate from differentiated electric organ expiants have not been observed to differentiate into electrocytes.Neuritic outgrowth in vitro occurs with electric lobe tissue taken at two different embryonic stages. The first stage corresponds to a period when most of the neuroepithelial cells in the lobe anlagen are withdrawing from the mitotic cycle and projecting axons into the branchial arches. The second, later stage is when the electromotorneurones are normally generating axon collaterals that are invading the interelectrocyte space of electrocyte columns. Maximum neuritic outgrowth at this second, later stage is obtained with tissue from 60–80 mm body-length embryos. Although neuritic invasion of electrocyte column expiants can be obtained in electric organ—electric lobe co-cultures at this later stage, synapses similar to those observed during the early stages of synaptogenesis in the electric organs in vivo have not been observed in vitro.     

Formation and stability of interpenetrating polymer network hydrogels consisting of fibrin and hyaluronic acid for tissue engineering

Fibrin gel is widely used as a tissue engineering scaffold. However, it has poor mechanical properties, which often result in rapid contraction and degradation of the scaffold. An interpenetrating polymer network (IPN) hydrogel composed of fibrin and hyaluronic acid–tyramine (HA–Tyr) was developed to improve the mechanical properties. The fibrin network was formed by cleaving fibrinogen with thrombin, producing fibrin monomers that rapidly polymerize. The HA network was formed through the coupling of tyramine moieties using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The degree of crosslinking of the HA–Tyr network can be tuned by varying the H₂O₂ concentration, producing IPN hydrogels with different storage moduli (G′). While fibrin gels were completely degraded in the presence of plasmin and contracted when embedded with cells, the shape of the IPN hydrogels was maintained due to structural support by the HA–Tyr networks. Cell proliferation and capillary formation occurred in IPN hydrogels and were found to decrease with increasing G′ of the hydrogels. The results suggest that fibrin–HA–Tyr IPN hydrogels are a potential alternative to fibrin gels as scaffolds for tissue engineering applications that require shape stability.     

Accelerated immunohistochemical staining by microwaves.

The application of microwave irradiation is extended to include the acceleration of the streptavidin-biotin peroxidase staining procedure for the detection of a wide range of antigens in paraffin-embedded sections of both normal and neoplastic tissues. Microwave irradiation is used for all procedural steps requiring incubation so that the entire staining procedure can be completed within 20 min. The technique is simple and convenient, with uniform irradiation times for different tissue antigens. The results obtained equalled that of conventional staining procedures.     

Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations.     

Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds

Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods that can take several days to obtain a final result, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for a more rapid and complete assessment. We report the development of a suite of real-time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically relevant, aerobic pathogens and demonstrates a high degree of sensitivity and specificity using a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared with those obtained by culture. As revealed by culture and PCR, the most common organisms were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the reference standard, respectively. The assays allowed the identification of bacterial DNA from ten additional organisms that were not revealed by quantitative or qualitative cultures. Under optimal conditions, the turnaround time for PCR results is as short as 4–6 h. Real-time PCR is a rapid and inexpensive approach that can be easily introduced into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by real-time PCR of chronic wounds is warranted.     

Removal of endogenous peroxidase activity from cryostat sections for immunoperoxidase visualisation of monoclonal antibodies

The interpretation of immunoperoxidase staining of frozen tissue sections can be severely limited by the presence of endogenous peroxidase enzymes in the tissue. Previously recommended methods of reducing this enzyme activity were examined, but were found to be unsatisfactory when applied to the small intestine. A method, which effectively eliminates this background staining but which does not interfere with the binding of monoclonal antibodies to various tissue components, is described. This method may prove useful in immunoperoxidase studies of other tissues in which high levels of endogenous peroxidase are present.     

An injectable,in situ enzymatically gellable,gelatin derivative for drug delivery and tissue engineering

A phenolic hydroxyl group was incorporated into gelatin, using aqueous-phase carbodiimide activation chemistry, to obtain in situ gellable and injectable protein-based materials for drug delivery and tissue engineering applications. By this means, gelatin derivatives that were gellable via a peroxidase-catalyzed reaction were obtained. The enzymatically cross-linked gelatin gels did not melt at 37 °C and showed tunable proteolytic degradability. The time necessary for gelation decreased with increasing content of the phenolic hydroxyl (Ph) group, peroxidase concentration and decreasing H₂O₂ concentration. Resistance to gel compression also depended on the content of Ph groups, with the gel containing the lowest Ph group content showing the greatest resistance to compression. We encapsulated L929 fibroblast cells in gelatin gels under conditions that induced gelation in about 10 s. The encapsulated cells showed about 95% viability. In addition, L929 cells seeded on the gels showed the same growth profiles as those seeded on an unmodified gelatin-coated dish. Subcutaneous rodent injection experiments demonstrated successful in situ formation of gels at the injected site.     

The relationship between brain tumor cell invasion of engineered neural tissues and in vivo features of glioblastoma

Glioblastoma is an aggressive brain tumor characterized by its high propensity for local invasion, formation of secondary foci within the brain, as well as areas of necrosis. This study aims to (i) provide a technical approach to reproduce features of the disease in vitro and (ii) characterize the tumor/host brain tissue interaction at the molecular level. Human engineered neural tissue (ENT) obtained from pluripotent stem cells was generated and co-cultured with human glioblastoma-initiating cells. Within two weeks, glioblastoma cells invaded the nervous tissue. This invasion displayed features of the disease in vivo: a primary tumor mass, diffuse migration of invading single cells into the nervous tissue, secondary foci, as well as peritumoral cell death. Through comparative molecular analyses, this model allowed the identification of more than 100 genes that are specifically induced and up-regulated by the nervous tissue/tumor interaction. Notably the type I interferon response, extracellular matrix-related genes were most highly represented and showed a significant correlation with patient survival. In conclusion, glioblastoma development within a nervous tissue can be engineered in vitro, providing a relevant model to study the disease and allows the identification of clinically-relevant genes induced by the tumor/host tissue interaction.     

Crosslinked collagen hydrogels as corneal implants: Effects of sterically bulky vs. non-bulky carbodiimides as crosslinkers

We have previously shown that recombinant human collagen can be crosslinked with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) to fabricate transparent hydrogels possessing the shape and dimensions of the human cornea. These corneal implants have been tested in a Phase I human clinical study. Although these hydrogels successfully promoted corneal tissue and nerve regeneration, the gelling kinetics were difficult to control during the manufacture of the implants. An alternative carbodiimide capable of producing hydrogels of similar characteristics as EDC in terms of strength and biocompatibility, but with a longer gelation time would be a desirable alternative. Here, we compared the crosslinking kinetics and properties of hydrogels crosslinked with a sterically bulky carbodiimide, N-Cyclohexyl-N′-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), with that of EDC. CMC crosslinking was possible at ambient temperature whereas the EDC reaction was too rapid to control and had to be carried out at low temperatures. The highest tensile strength obtained using optimized formulations were equivalent, although CMC crosslinked hydrogels were found to be stiffer. The collagenase resistance of CMC crosslinked hydrogels was superior to that of EDC crosslinked hydrogels while biocompatibility was similar. We are also able to substitute porcine collagen with recombinant human collagen and show that the in vivo performance of both resulting hydrogels as full-thickness corneal implants is comparable in a mouse model of an orthotopic corneal graft. In conclusion, CMC is a viable alternative to EDC as a crosslinker for collagen-based biomaterials for use as corneal implants, and potentially for use in other tissue engineering applications.     

Protective role of curcumin in nephrotoxic oxidative damage induced by vancomycin in rats

Vancomycin (VAN) is a glycopeptide antibiotic which is active against gram positive bacteria including methicillin resistant Staphylococci. Free radicals are involved in the pathogenesis of vancomycin-induced nephrotoxicity. Therefore, the present study was conducted to investigate the antioxidant potential of curcumin (CUR) against the nephrotoxicity of vancomycin in male rats. Animals used in this study were divided into four groups; the first group was used as control, the second, third and fourth groups were treated orally with curcumin (200 mg/kg BW/day), vancomycin (200 mg/kg BW/day, i.p.), vancomycin plus curcumin, respectively. Curcumin was administered 2 weeks before and 1 week simultaneously with vancomycin. Results showed that thiobarbituric acid reactive substances (TBARS) in plasma and kidney tissue were significantly increased after vancomycin administration. While, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in plasma and kidney tissue and the content of glutathione (GSH) in kidney tissue were decreased compared to control. Vancomycin significantly increased the levels of urea and creatinine. The presence of curcumin with vancomycin caused reduction in induction levels of TBARS in plasma and kidney, urea and creatinine. It ameliorated vancomycin-induced decrease in the activities of antioxidant enzymes and GSH. The presence of curcumin with vancomycin alleviated its nephrotoxic effects. It can be concluded that curcumin has beneficial influences and could be able to antagonize vancomycin nephrotoxicity.     

Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection

We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee (n = 272) or hip (n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture (P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure.     

Binding and inhibition of myeloperoxidase (MPO): a major function of ceruloplasmin?

Interactions between plasma proteins and MPO were studied. The protein fraction of normal plasma and serum was shown to exhibit an inhibitory effect on the peroxidase activity of MPO. Most of the inhibitory effect could be retained on an MPO-coupled affinity chromatography column. In particular, a protein with apparent mol. wt of 130 kD showed affinity for MPO. The protein was identified as ceruloplasmin by N-terminal amino acid sequencing and immunochemistry. During separation procedures the peroxidase inhibitory effect was limited to ceruloplasmin-containing fractions of plasma. Purified ceruloplasmin inhibited the peroxidase activity of MPO in a concentration-dependent manner, and exhibited selective binding to MPO-coated microtitre plates. This binding could be inhibited by MPO dissolved in buffer. Correspondingly the binding of MPO to ceruloplasmin-coated plates could be blocked by ceruloplasmin in solution, showing a physical interaction to occur between the two proteins under physiological conditions. We also found affinity to exist between MPO and C3 (and its C3d-containing fragments). However, C3 and C3 fragments did not inhibit the peroxidase reaction in vitro. We propose that ceruloplasmin takes part in the clearance and inactivation of MPO, in vivo.We also speculate that impaired inactivation of MPO may have a pathophysiological role in inflammatory diseases characterized by autoantibodies to MPO, such as rapidly progressive glomerulonephritis with P-ANCA (perinuclear anti-neutrophil cytoplasmic antibodies).     

Expression of pendrin and NIS iodide transporters in human breast tumor and peri-tumoral tissue

IntroductionThyroid iodide transporters, Na⁺/I^– symporter (NIS) and pendrin (PDS), are responsible for supplying this vital micronutrient for thyroid hormone synthesis by thyroid peroxidase (TPO). Both proteins were shown to be expressed, apart from the thyroid, also in other human tissues, including lactating mammary gland. NIS expression in human breast cancers has been widely studied. On the other hand, until now PDS mRNA levels in breast tumor tissue have been estimated only in high throughput analyses. Previously, we have observed that TPO is expressed in normal and cancerous human breast tissues and shows enzymatic activity. However, biochemical activity of TPO in human breast cancer cells requires iodide transport by NIS and PDS. Therefore, to extend our previous study on TPO expression and function in human breast tumors we performed analysis of NIS and PDS levels in the same group of patients.Material and methodsThe study involved detection of NIS and PDS protein levels by immunohistochemistry and Western blotting, as well as mRNA levels by real-time quantitative polymerase chain reaction.ResultsHere we provide direct evidence that NIS and PDS are expressed in human breast cancer tissue, with NIS levels being increased and PDS levels decreased in tumor tissue. Interestingly, PDS mRNA levels in breast cancer tissue seem to be influenced by the estrogen receptor status and age of the patients, while NIS mRNA levels were dependent on histological type of the tumor.ConclusionsThis study provides valuable information important for consideration in diagnostic or therapeutic application of radioiodine in breast cancer management.     

Evaluation of the Antioxidant Effects of Ziziphus Mauritiana Lam. Leaf Extracts Against Chronic Ethanol-Induced Hepatotoxicity in Rat Liver

Chronic alcohol ingestion is known to increase the generation of reactive oxygen species (ROS), thereby leading to liver damage. Antioxidant enzymes act individually or in combination to reduce or counter the effect of these ROS. Chronic administration of alcohol at (40% v/v, 1ml/100g), for 6 weeks showed a significant (p<0.05) elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TB). There was also a significant (p<0.05) decreased levels of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase compared to control rats. Pre-treatment of rats with 200, 400 mg/kg body weight of aqueous leaf extract of Ziziphus mauritiana or 100 mg/kg silymarin resulted in a significant (p<0.05) decreased levels of ALT, AST, ALP, and TB with levels of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase showing a significant (p<0.05) increase compared to group administered alcohol only. Histopathology of rat liver administered with alcohol only resulted in severe necrosis, mononuclear cell aggregation and fatty degeneration in the central and mid zonal areas which was a characteristic of a damaged liver. Pre-treatment with the aqueous extract of Ziziphus mauritiana or silymarin reduced the morphological changes that are associated with chronic alcohol administration. The presence of tannins, saponins and phenolic compounds observed in the plant extract could be responsible for the observed effects of decreasing the levels of injured tissue marker and lipid peroxidation.