INTRODUCTION:

Ki67 is a type of nuclear antigen protein of a cell, which is usually detected by monoclonal antibody Ki-67 in the all phases of cell cycle except in the non-proliferating phase or G_Ophase¹. This give the Ki67 function as useful in the experiment to differentiate the dividing cells from the non-dividing cells group.

The correlation of Ki67 and [³H] thymidine cell expression during S phase of cell cycle added to its suitability marker to be used in a cell proliferation test². Furthermore, Ki67 is widely used in the staging of the tumour and study of cancer. For example in cancer cell test, there was a correlation of immunoperoxidase Ki67-stained cells with the histological staging of brain tumour germinoma and cerebral lymphoma biopsies which used to identify cancer cells³. The changing trend of [³H] thymidine usage into 5’-bromodeoxyuridine (BrdU) prompt it to be used in identifying the S phases of cancer cells such as in human breast cancer cells⁴. [³H] thymidine is known for the difficulty in its technique compared to BrdU. Since the emergence of BrdU, Ki67 was shown of co-labelling with BrdU in the dentate gyrus of rat, where the dentate gyrus brain area is a known area of neurogenesis⁵.

Functionally, Ki67 protein is arguably one of the essential proteins of cell proliferation⁶. This is explained by the higher of Ki67 labelling index in the ependymal of adult spinal cord of injured rat compared to uninjured of similar animals⁷. In addition, Ki-67 is speculated belongs to a family of signal transduction protein group linked to cell cycle phases⁸. These facts give the Ki67 as good marker for dividing or proliferation process of the cell.

Ki67 is viewed as better indicator than other proliferation markers such as BrdU based on its inability to induce stress of injection⁵. However it is not suitable for long period tracing of cell division products through co-labelling method⁵.

In an experiment identifying the acid-sensing cells, PKD2L1-expressing cells or acid-sensing cells are found both in the tongue and around central canal of spinal cord, where these cells are believed detect the acid changes around these organs⁹. Since the similar existence of Ki67 around neurogenic area such as central canal of the spinal cord⁷, it is speculated Ki67 cells are also presence in the oral cavity which may be exist there. Here in this research, Ki67 cells are mostly found in several part around oral region.

MATERIALS AND METHODS:

Adult Sprague Dawley rats were used in this experiment. Ki67 (1:200) Invitrogen, USA.

The animals were deeply anaesthesized with Pentobarbitone Sodium 20% w/v (60mg/kg) which administered intraperitoneally, pedal reflexes are checked to verify that the animal is fully sedated, the abdomen dissected transversely with a surgical scissor then a thoracotomy performed to access the heart. The animals were perfused with 200ml 4% Paraformaldehyde (PFA) in 0.1M Phosphate buffer (PB) with pH 7.4. The spinal cord and tongue were removed and cut in horizontal section. Then all the sections were immersed and fixed in 10% formalin solution for 24 to 48 hours.The spinal cord and tongue were placed in individual labelled cassette. for the overnight (13 hours).

The processed slides underwent preliminary treatment for paraffin sections and with TrisEDTA buffer pH 9. About 2-3 drops of Peroxidase Blocking agent were placed into the slides to neutralize the endogenous peroxidase activity. The slides were then incubated for 5 minutes. Rinse the slides with distilled water followed by TBS solution. Then the primary antibody (anti-Ki67 diluted at 1:200) were applied to the slides and were incubated for 30 minutes at room temperature.

After that, the slides were washed twice with TBS solution. Two to three drops of secondary antibody (Horseradish peroxidase) were then applied to the slides and were incubated for 30 minutes at room temperature. Then, the slides were washed twice with TBS solution. The slides were taken out from immunostainer and were placed in TBS solution. Then, the slides were flooded with DAB for five minutes at room temperature. After that, the slides were washed thoroughly under running tap water.

Counterstain were done by immersing the slides into Harris Haematoxylin for five seconds. This was followed by washing the slides under running tap water (bluing process).

The slides were immediately processed for dehydration after the staining process was done. This was carried out by bringing the slides to increasing concentration of alcohol from 70% and 80% alcohol to two changes of 95%alcohol and two changes of absolute alcohol with a minute each. The slides were then cleared in three changes of xylene for a minute each before they were finally mounted with DPX mounting media and cover slipped. The slides were viewed under the light microscopy. Images then were taken using image analyser and be analysed and summarized. The Ki67-expressing cells are the cells that were stained with dark-brownish colour.

RESULTS:

Distribution of Ki67 expressing cells in the spinal cord:

The spinal cord of a rat was divided into several slices into 4 regions: cervical, thoracic, lumbar and sacral. The middle part of each sections was chosen for the staining to prevent overlapping of each regions.

To determine the area that contain proliferating cells marked by Ki67 in the spinal cord, first the area that known to contain proliferating cells which is the spinal cord specifically Lamina X area of its gray matter was identified. Internally, spinal cord contains two areas- white matter and grey matter, where grey matter is located inner than white matter. Lamina X is an inner area of gray matter of the spinal cord which surround the central canal. Here, the proliferating cell can be found in the ependymal cells area and surrounding the central canal as similar as shown by the experiment using BrdU (10). The spinal cord of the rats was taken and the middle part of each segment from cervical, thoracic, lumbar and sacral were chosen. Each of the part were further cut into slices using microtome into small slices.

In this experiment, the Ki67 expressing cells are found in around middle area of spinal cord, which is known as Lamina X of the gray matter of the spinal cord (Figure 1). The cells are identified via brownish colour staining in contrast with surrounding blue-coloured cells by DAPI (Figure 1 and 2). No other area of the spinal cord – white and gray matter laminas were noted containing these Ki67 cells.

The number of the cells are quite small (average 1 or 2 Ki67 cells) per slices of the tissue. The slices were 2 µm in length. The morphology of the cells was similar with the bluish non-Ki67 ependymal cells surrounding them- oval in shape. There were no processes seen come out via these cells. The size of the Ki67-cells were estimated around 5 to 10µm.

Figure 1. The Ki67 expressing cells in the spinal cord and tongue. Ki67-expressing cells (arrows) in the Lamina X of grey matter at the lumbar level of the spinal cord (40x magnification) (A). Ki67-expressing cells in the taste buds of the tongue, at 4x magnification at their bases (B). Ki67-expressing cells (arrow) at the base of taste buds, at 40x magnification. Note the papilla or apex of the taste buds were not stained (C).

Distribution of Ki67 expressing cells in the oral region (tongue):

To determine the area around oral region that may contains Ki67 expressing cells, we focused the searching in the tongue as it was shown to contains PKD2L1-expressing cells- which speculated to be acid-sensing cells (9). The tongue was cut into several part (transverse section) and its middle part were taken for the testing. Here the tongue sections contain several structures such as taste buds. The other structures that can be visualised together with the tongue sections were bundle of nerve fibers, a group of neurons and thyroglossal duct.

The Ki67 expressing cells were found at the area of the base of taste buds. The taste buds were found in the dorsal area of the section, which were identified by papillary-like structure surrounded by several layers of cells marked by DAPI. The base cells were stained with brown compared to bluish coloured DAPI of non-Ki67 cells. The stained Ki67 cells of the tongue were similar to the spinal cord in various aspects. The stained-cells colour were range from mild to dark staining (Figure 1). The morphology of the Ki67 cells were oval in shape similar to the shape with the non-Ki67 cells around them. The cell size was approximately around 5 to 10µm in diameter.

The taste bud cells located at the papilla region of the bud were not stained.

Distribution of Ki67 expressing cells in the oral region (thyroglossal duct):

Through the transverse cut of the middle tongue, the structure containing Ki67 expressing cells are thyroglossal duct. The thyroglossal duct can be found located in the ventral area to the tongue. The thyroglossal duct can be identified by a cavity surrounded by the several cells marked by the DAPI. Majority of the cells of surrounding the thyroglossal duct were stained with Ki67 marker (Figure 2). However, some of it were not stained. The colour of the stained Ki67 cells were brownish in colour and the size was approximately 5 to 10µm in diameter. The morphology is similar with the Ki-67 cells of the taste bud which is oval and round in shape.

Distribution of Ki67 expressing cells in the oral region (ganglion).:

In addition, the structure containing Ki67 expressing cells is a group of cell bodies which possibly is a ganglion. The ganglion is located in the ventral area to the tongue region. The ganglion is identified via its structure containing several cell bodies of neurons. The neurons in the ganglion are stained with brownish colour of Ki67 (Figure 2). The size of the neurons are approximately 5 to 10µm and no processes can be seen from the neurons.

There were few bundles of axons of nerve fibres were found at the ventral area to the tongue. There bundle of these axons were not stained with Ki67.

Figure 2. Ki67-expressing cells around oral cavity of mammals. Ki67-expressing cells (arrows) in the thyroglossal duct, at 20x magnification (A). Ki67-expressing cells (arrows) in the thyroglossal duct, at 40x magnification (B). Ki67-expressing cells (arrows) in the ganglion containing neurons, at 40x magnification (C). The axons of nerve fibres of the tongue which are not stained by Ki67, at 40x magnification (D).

DISCUSSION:

Distribution of Ki67 expressing cells in the spinal cord:

The Ki67 expressing cells were found in the area of the gray matter of the spinal cord known as Lamina X. The cells were found in the ependymal area which surround the central canal of the spinal cord throughout spinal cord from cervical, thoracic, lumbar and sacral spinal cord. This strengthening previous results¹⁰ that Lamina X of the rat contains cells capable of undergone cell cycle stages in the normal condition.

The cells were found in small quantity (one or two) per slices in this experiment shows only small quantity of cells capable to divide themselves in a normal condition. The majority of the cells which were not marked by the Ki-67 might be capable to be stained in certain condition such as tissue injury⁷. The shape of the Ki67 cells shows it is in the form of similar with the ependymal cells of the Lamina X in which they are not differentiated into any other cells yet.

Compare to previous result¹⁰ they found the existence of other areas of spinal cord containing proliferating cells whereas in this experiment we found only one area- Lamina X area. This shows the differences of cells undergoing cell cycle stage in the normal condition where Lamina X contains cell that going to full cycle of cell cycles by expressing Ki67 proteins while other cells in other areas of spinal cord specifically stays in S phase of cell cycle evidenced by BrdU expressions¹⁰. The difference marked the difference in using the marker in the experiment, where Horner et al used BrdU which incorporation of uridine into DNA in S phase of cell cycle in comparison with our experiment using Ki67 which detecting the protein expressed during all phases of cell cycle. The incorporation of uridine into S phase sense the changes of DNA itself whereas the Ki67 markers shows both changes of the S phases and the division of the cell itself.

Distribution of Ki67 expressing cells in the tongue.:

In the tongue, the Ki67 expressing cells were found in the taste buds region specifically at their bases. This indicate the capability of the cells at the base to undergone cell cycle phases. The Ki67 protein are expressed in the normal condition at the base shows the cells are very active whether in dividing stage or in the stage of DNA changes of S phase. The cells in the papilla part of the taste bud does not show the Ki67 protein expression could shows the cells are in not in the cell cycle. This could speculate that the cells at the bottom of the taste bud actively divided during normal condition to replace the cells at the top of the taste buds.

Based on Huang et al findings, the existence of PKD2L1-expressing cells of the taste bud could possibly support the detection the acidic changes of the activity from the base part of taste bud⁹.

Distribution of Ki67 expressing cells in the thyroglossal duct and ganglion:

The Ki67 expressing cells in the group of cell bodies of the neuron ventral to the tongue could indicate the neurons may capable to express the Ki67 proteins during normal condition. Since the neuron is usually not known to divide itself, it may indicate the changes of DNA possibly the repair during normal condition.

The existence of Ki67 proteins in the thyroglossal duct cells may indicate the activity of the cells of undergone cell cycle in the normal condition. This activity of the cells may have clinical value.

The absence of Ki67 in the axons at the ventral part of the tongue show the incapability of the axon to show Ki67 proteins since they are not contain the DNA.

CONCLUSION:

The Ki67 cells were identified in oral region organs in the cells of the base of the taste bud of tongue, thyroglossal duct and a group of ganglion containing neuron cell bodies.

ACKNOWLEDGEMENT:

The authors wish to extend their appreciation to Universiti Sains Malaysia (USM) for generously funding this study via The Short Term Research Grant (STG): 304/PPSG/61313108.

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Received on 17.01.2025 Revised on 22.03.2025

Accepted on 30.05.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2860-2863.

DOI: 10.52711/0974-360X.2025.00411

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