This study was part of a larger research project and it adopted case control as the experimental research design. Two (24.6kg and 25.2kg) domestic pigs ( Sus scrofa Linnaeus) were acquried from veterinary swine center at Kabete, Kanyariri, Kiambu County, Kenya. One of the pigs was used as a control and the other was experimental.

The pigs used in this study were issued by the University of Nairobi Faculty of Veterinary Medicine veterinary farm, where animals are specifically bred and maintained for research and training purposes. As such, no private ownership was involved, and institutional authorization was granted for their use in accordance with ethical and welfare regulations.

Ethical approval for animal use was provided by Biosafety, Animal Use and Ethics Committee of the Department of Veterinary Anatomy and Physiology, Faculty of Veterinary Medicine, University of Nairobi. REF: FVM BAUEC/2019/203.

The study was carried out in the upper Kabete region of Kiambu County, situated within the veterinary farm of the University of Nairobi (Kenya). The study site was a soft wood tree forest characterized by a black soil and low tree cover. The site was located at an altitude of 1900m a.s.l, representing a typical montane region.

Two pigs (Sus Scrofa L.) weighing 24.6 and 25.2kg each were used as human models to determine effect of flunitrazepam on the decomposition of liver in an open area of thickest forest at the upper Kabete campus farm, University of Nairobi, Kenya in the month of July 2021 (16 Days). One pig was used as a control and the other one as an experimental pig. Both pigs were allowed a three-day acclimatization period to adapt to the environmental conditions before the study commenced. On the evening of June 30, 2021, the experimental pig was administered 2 mg of flunitrazepam dissolved in 250 ml of vodka (40% ethanol composition). On the morning of 1st July, 2021, at 5:00 a.m., the pigs were euthanized using an electric stunning method which involved applying an electric current through the brain. This is a commercially allowed method in slaughterhouses in Kenya by the Directorate of Veterinary Services in protecting the welfare of animals to ensure a humane and rapid procedure. The abdomen was then opened to expose the liver for sampling the then placed on an open ground and secured against preditors with metal cages as shown in Figure 1.

Small pieces of liver tissue samples were obtained from both control and experimental animal daily and preserved in 15 ml of 10% formalin (Sup: Loba chemie, Cat No: 00146) per sample from day 1 to day 16 as shown in Figures 2 and 3 respectively. The control and experminetal slides were labelled control histopathology day (CHD) and experimental histopathology day (EHD) (From day 1 to day 16) respectively. These tissue samples were then processed to assess histopathological changes and the impact of flunitrazepam on the decomposition process. The changes occurring at both tissue and cellular levels were observed using an electron microscope to determine the degree of autolysis/decomposition over time, and images were captured along with descriptions.

Following fixation, the liver tissues were routinely processed for sectioning according to protocol by Gunasegaran. ¹¹ The samples were placed in a glass slide in 10% neutral buffered formalin (NBF), (pH = 7.0) and allowed to fix flat. These samples were then placed in 2.0 ml snap cap eppendorf tubes in 10% NBF and fixed at 4 °C for between 16–24 hr. Following fixation, samples were rinsed in running tap water overnight then dehydrated in 250 ml ascending concentrations of ethanols (50%, 70%, 80%, 90%, 95% and 100%) (sup: Scharlau, Cat No: ET00052500). ¹² The samples were then cleared in two changes of methyl benzoate, infiltrated and embedded in molten paraffin wax then left to solidify overnight. Blocking was done by attaching the wax embedded samples onto wooden blocks. Tissue sections 5 to 7 μm thick were then cut from the embedded blocks 13322 a leitz Weitzar ® rotary microtome. ¹³ The sections were then mounted on glass slides, deparaffinised using 250 ml of xylene (Sup: Loba Chemie PVT LTD, Cat No: 00370), rehydrated in a 250 ml through decreasing concentrations of ethanol (100%, 90%, 70% and 50%) and finally in distilled water. ¹⁴ The rehydrated sections were then subjected to staining protocol; Hematoxylin & Eosin (H&E) and Masson’s trichrome staining (Sup: Loba Chemie PVT LTD). The stained sections were then examined and photographed using a Leica ® DM 500 light microscope.

Day One

The main structural appearance of the liver in day one are illustrated in Figure 4, liver tissue of the control specimen showed an intact cuboidal epithelium of bile ducts on the basement membrane within the portal tract in the first day. Furthermore, the endothelium of hepatic artery and portal vein were intact, and the portal connective tissue was well-organized with evidence of portal congestion. ¹⁵ In the lobules, the lobular integrity was preserved, and hepatocytes appeared intact with a visible nucleus. The sinusoids were distended with red blood cells (RBCs). Additionally, a few hepatocytes displayed early stages of autolysis, characterized by the loss of details, such as cytoplasmic boundaries.

Histopathological features of the liver in experimental pig are illustrated in Figure 5. Similar to the control, the experimental liver tissue exhibited an intact cuboidal epithelium of bile ducts on the basement membrane within the portal tract. The hepatic artery endothelium and portal vein endothelium were also intact, and evidence of portal congestion was observed, with the portal connective tissue being well-organized. ¹⁶ The lobular integrity was preserved, with intact hepatocytes displaying a visible nucleus and distended sinusoids filled with red blood cells. Scattered hepatocytes in the lobules showed early stages of autolysis, characterized by a loss of details, such as cytoplasmic boundaries. However, the experimental liver also exhibited large foci of lost hepatocellular details, indicating more advanced stages of autolysis.

Day Two

In Day two, as shown in Figure 6, the portal tract lesions in the control specimen replicated those of Day one. However, the lobules showed loss of cell boundaries, leakage of cytoplasm and free nuclei trapped in eosinophilic coagulum of leaked cytoplasm.

In the experimental specimen, as illustrated in Figure 7, the liver tissue within the portal tract exhibited intact blood vessels, bile ducts, and connective tissue. However, pools of red blood cells were observed outside the blood vessels, indicating hemorrhage. Furthermore, the lobular pattern was mainatained and the lobules displayed a loss of hepatic columns with individualization of hepatocytes and lost integrity of sinusoids and borderless pools of red blood cells, indicating further hemorrhage. The hepatocytes showed individualization with a loss of cell-cell boundaries, with the majority exhibiting a clear cell structure and boundaries. However, nuclear pyknosis was frequently observed, along with occasional instances of nuclear abnormalities.

Day Three

On day three, as shown in Figure 8, the control specimen portal tract lesions remained similar to those observed on day one, with intact connective tissue, arterial and venous structures, and congested blood vessels. The bile duct epithelium remained intact, maintaining its orderly cuboidal structure on the basement membrane, although slight cellular swelling was noted. Furthermore, the lobular pattern remained intact, but the hepatic columns were absent, and there was a loss of cell-to-cell boundaries with a predominance of ghost hepatocytes. The sinusoids showed distension with red blood cells, and nuclear pyknosis was observed in scattered hepatocytes, indicating progressive autolytic changes. ¹⁷ The results also revealed prominent nucleus changes, such as pyknosis, and individualized hepatocytes, with some clumped into multinucleated hepatocytes. The loss of sinusoidal arrangements was also observed, along with the occurrence of brown pigments (bile pigments), RBC swelling, and hemolysis.

The Histopatology of liver in experimental pig on day three are illustrated in Figure 9. Mainly, the portal tract was intact, with distended veins filled with blood, intact connective tissue, and bile ducts. The lobular pattern was preserved, but the hepatic columns were lost, leaving ghost-like remnants of dead cells. Furthermore, a pink coagulum of leaked cytoplasm was observed in the lobules, along with prominent distension of sinusoids with red blood cells. Scattered free bile pigment was also observed, and the majority of the red blood cells appeared swollen and mostly hemolyzed.

Day Four

On day four, as shown in Figure 10, the portal tract in control specimen exhibited a lack of bile ducts, and the blood vessels were shells that lacked a clear endothelial lining. Additionally, the arterial blood vessel wall had loose spongy tissue of non-viable cells, and there was a loss of connective tissue, leaving empty spaces in the wall. In the lobules, the hepatocyte columns were lost, and there were loosely sequestrated clumps of ghost hepatocytes, which were elongated in shape. Furthermore, there was no evidence of red blood cells and hepatic sinusoids were indistinct. The structural changes were extensive and widespread throughout the lobules.

Histological features of liver in experimental specimen on day four are illustrated in Figure 11. The portal tract displayed shells of blood vessels with non-viable cells and a lack of clear endothelium Furthermore, no evidence of red blood cells was observed within the lumen, and the bile ducts were not evident. The connective tissue framework around the lobules was reduced, with areas of absence, causing adjacent lobules to collapse into each other. In the lobules, the hepatic column was lost, and there were no viable hepatocytes or sinusoids. Amorphous to elongated clumps of ghost hepatocytes were observed, along with pink-orange pools of clumps of hemolyzed red blood cells.

Day Five

In the control specimen, as shown in Figure 12, the liver tissue’s portal tract exhibited shells of blood vessels, but these structures lacked viable cells and showed no evidence of bile ducts or connective tissue. The bile duct epithelium was absent, and the remnants of the blood vessels were surrounded by loosely organized debris. Within the lobules, there was a complete absence of interlobular connective tissue, resulting in a loss of the characteristic lobular pattern. Sinusoids and red blood cells were no longer visible, reflecting advanced autolytic changes. The hepatocytes were represented by enlarged clumps of ghost cells, with no discernible parenchyma or cellular details. These findings highlight a significant progression in tissue degradation compared to earlier days, characterized by the near-total breakdown of structural integrity and cellular components.

In the experimental liver tissue portal tract, the blood vessel “shells” were non-viable, and there were no bile ducts or viable connective tissues ( Figure 13). In the lobules, minimal interlobular non-viable connective tissues were observed, with surrounding lobular patterns being non-viable, and no evidence of sinusoids or intact hepatocyte columns. ¹⁸ The results indicated the presence of non-viable, amorphous clumps of ghost hepatocytes, with shadows of nucleus and cytoplasm. Furthermore, no visible parenchyma was observed between hepatocytes, and there was no evidence of red blood cells, with isolated pink-orange pigments indicating hemolyzed red blood cells.

Day Six

On day six, as shown in Figure 14, the control tissue’s results remained consistent with those observed on day five (CHD5), with the addition of blue/basophilic staining clumps observed in the blood vessels, which were consistent with bacterial colonies.

The results, as shown in Figure 15, indicated that in the portal tract of the experimental liver tissue, the blood vessels were without a viable endothelium, the bile duct was lost, and the portal connective tissue was lost. In addition, the interlobular connective tissue was lost, there was no lobular pattern, no sinusoids and no RBCs in blood vessels and the amorphous hepatocytes were separated by empty spaces (no parenchyma).

Day Seven

The results, as shown in Figure 16, show that in the portal tract of the control tissue on day seven, there were shell of blood vessels, many artifacts, no RBCs in the vessels, no bile ducts and no connective tissue. The results also show that there was no lobular pattern, no interlobular connective tissue, there were dead individualized hepatocytes, there were large empty spaces separate hepatocytes (no parenchyma) and the shape of ghost hepatocytes was polymorphic.

The findings revealed that in the experimental specimen liver tissue portal tract, the blood vessel wall remained intact, with clumped red blood cells and dark blue clumps in the lumen consistent with bacterial colonies ( Figure 17). ¹⁹ No evidence of bile ducts was observed, with debris present in the lumen. Concerning the lobules, the interlobular demarcation of lobules by fibrous tissue was present, with non-viable hepatocytes, individualized ghosts of hepatocytes, and a lack of hepatic columns.

Day Eight

On day eight, the examination of the control liver tissue portal tract revealed the presence of non-viable shells of blood vessels, with no bile ducts observed ( Figure 18). Moreover, the interlobular connective tissue was lost, and the hepatocytes had undergone necrosis, resulting in individualized ghost cells with no observable hepatic columns.

As illustrated in Figure 19, the examination of the experimental specimen liver tissue portal tract showed the presence of non-viable shells of blood vessels, with no evidence of bile ducts observed. Additionally, there was a loss of interlobular connective tissue, resulting in hepatocytes undergoing necrosis and the formation of individualized ghost cells with no observable hepatic columns.

Day Nine

In the portal tract of the control’s liver tissue on day nine, there were shell of blood vessels, there were no bile ducts, there was loss of interlobular connective tissue, the hepatocytes necrotized and there were individualized ghost cells (no hepatic columns) ( Figure 20). In addition, there was collapse of wall of arterial blood vessels diminishing the luminal volume sometimes to total absence.

As demonstrated in Figure 21, the outcomes indicated that in the experimental tissue, there were clumps of amorphous eosinophilic masses of non-viable hepatocytes. Furthermore, the contours of blood vessels with clear lumens were observed, and the interlobular connective tissue was disintegrating, resulting in shells of empty spaces in between. Additionally, there were individualized ghosts of scattered hepatocytes separated by conspicuous empty spaces, with the density of ghost hepatocytes being less than in EHD8. The dissolution at EHD9 was more noticeable.

Day Ten

On day 10, as indicated on Figure 22, observations showed that blood vessels on the control’s liver tissue had contracted. The connective tissue among the interlobules had dissolved but not bile ducts, therefore, providing a ghost hepatocytes appearance.

The results revealed that in the experimental liver tissue on day 10 ( Figure 23), there was a complete loss of hepatocytes, and the ghost hepatocytes were more densely packed with no discernible individualization. Furthermore, the interlobular connective tissue had disintegrated, leaving empty shells. ²⁰ Additionally, the results showed that dark blue clumps were present in the lumen of the ghost blood vessels, which indicates the presence of bacteria as determined by staining.

The study findings revealed that on day 11, the liver tissue of the control exhibited clumped eosinophilic masses, loss of interlobular connective tissue, clumps of necrotic cells, and absence of blood vessels and bile ducts. ²¹ Additionally, the results indicated the presence of amorphous clumps of dead tissue with varying degrees of eosinophilic staining, dispersed within noticeable empty spaces.

The findings were that on day 11, no evidence of connective tissue, vessels, and bile ducts was seen. ²² The findings also revealed that there were groups of dead tissue (eosinophilic) and big/profuse clusters of blue staining masses in ghost of vessels.

On day 12, the results revealed that the liver tissue of the control specimen displayed shells of blood vessel walls, debris, and empty spaces. Furthermore, there were multifocal basophilic staining masses distributed in the shells of blood vessels. ²³ The interlobular connective tissue had completely dissolved, resulting in a loss of tissue eosinophilic staining.

On day 12, the experimental specimen exhibited significant findings. The liver tissue showed eosinophilic staining with dissolved interlobular connective tissue and extensive tissue loss. Moreover, the remnants of blood vessel walls were observed, albeit devoid of any blood cells. The bacterial colonies were identified through basophilic staining. Additionally, all hepatocytes were determined to be dead, leaving behind only ghost remnants.

On day 13, the liver tissue of the control specimen exhibited loss of tissue eosinophilia, coagulum of dead tissue, absence of interlobular pattern and connective tissue, and lack of blood vessels/traces. The results also showed a significant occurrence of multifocally distributed basophilic masses (bacterial colonies) not clearly marked by vessels boundaries. Furthermore, there were conspicuous empty spaces in the tissue.

Similarly, experimental tissue within the specimen exhibited loss of tissue eosinophilia, necrotic tissue coagulum, absence of interlobular pattern, absence of connective tissue, and absence of vessels/traces. Additionally, there were many empty spaces, but unlike with the control specimen, there were no multifocally distributed basophilic masses (colonies of bacteria) with indistinct vessel boundaries.

On Day 14, control specimen liver tissue exhibited characteristics as on Day 13, including tissue eosinophilia loss, absence of interlobular pattern, absence of connective tissue, and absence of vessels/traces. Still, there were a number of empty spaces and few stained basophilic masses (multifocally distributed; bacterial colonies). Furthermore, tissue debris was separated by empty spaces and there was no visible eosinophilia.

The liver tissue in the experiment specimen on day 14 was characterized by scattered eosinophilia of dead tissue, tissue debris separated by empty spaces, no basophilic masses and no connective tissue or blood vessels. On day 14, the liver tissue in the experimental specimen exhibited scattered eosinophilia of dead tissue with tissue debris separated by conspicuous empty spaces. Notably, there were no basophilic masses, connective tissue, or blood vessels present.

On day 15, the liver tissue in the control specimen demonstrated the presence of silhouettes of blood vessels with basophilic staining masses (bacterial colonies), scanty eosinophilia, and tissue debris separated by conspicuous empty spaces. Furthermore, there was a loss of interlobular connective tissue, resulting in the absence of the characteristic lobular pattern.

On Day 16, the liver tissue in the control specimen was similar to that of Day 12, characterized by multiple empty spaces separated by debris, multifocally scattered basophilic masses (bacterial colonies), and a lack of eosinophilia. On day 16, the liver tissue in the experimental specimen exhibited clumped masses of dead tissue with conspicuous empty spaces due to autolysis and complete loss of tissue architecture. The tissue also showed coagulum of eosinophilic debris with numerous empty spaces, while the clumped basophilic masses were absent, indicating the absence of bacterial colonies.

From the results, as shown in Figure 24, EHD1, EHD2, EHD3, EHD4 and CHD1, CHD2 show normal liver tissue with normal architecture. The hepatocytes arranged in plates, polygonal cells with round, centrally located nuclei and abundant eosinophilic cytoplasm with fine basophilic granules; are pleomorphic and multinucleated. ²⁴ Kupffer cells are located in the sinusoids and are mononuclear; they are phagocytic cells (macrophages) that respond to cell injury. ²⁵ The cells are in bean shaped nucleus with abundant cytoplasm and star shaped cytoplasmic extensions. Endothelial cells lining sinusoids as well as blood vessels with indistinct cytoplasm and small elongated nuclei were observed.

CHD3 demonstrates the ratio of liver tissue organization is preserved at 70%, and hepatocytes along with Kupffer cells and endothelial cells are also identified. CHD4 and EHD5 indicate that the architecture of the liver tissue remains at 60% and hepatocytes, Kupffer cells, and endothelial cells are visible. CHD5 and EHD5 indicate that the architecture of liver tissue is retained at 50% while hepatocytes, Kupffer cells, and endothelial cells are visible. CHD6 and EHD6 indicate that 45% of liver tissue architecture is maintained and hepatocytes, Kupffer cells, and endothelial cells are presented. CHD7 and EHD7 retains the liver tissue organization at 35%, and hepatocytes, Kupffer cells, and endothelial cells are present.

The hepatocyte arrangements of the liver are preserved at 30% while the cellularity of the tissue is distorted at 80%. CHD9 and EHD9 depict that the porosity of the liver tissue architecture is at 25% and the cellularity is altered at 85%. CHD10 and EHD10 exhibited a fully disrupted structure of the tissue along with ghost cells. The total tissue, as shown in CHD11, CHD12, CHD13, CHD14, CHD15, CHD16, EHD11, EHD12, EHD13, EHD14, EHD14, EHD15 and EHD16, is unrecognizable.