INTRODUCTION:

Several techniques are being used for the color visualaization of animal cartilage and bone structures in biology, anatomy and embryology. One among them is the diaphonization method. Diaphonization is the preservation technique in which the tissues of a specimen is made transparent and the cartilage and the bones are made visible by using specific dyes. This method is based on the permanent binding of some dyes with bone calcium and selective binding of other dyes with cartilage structures of biological specimens⁽¹⁾. Diaphonization comprises consecutive fixation of biological specimens in formalin, rehydrating in ethanol, digesting in trypsin, staining with the respective dyes and preserving in glycerin. Initially diaphonization was developed by Schultze in the year 1897⁽¹⁾.

Later many it was modified by many researchers like Mall (1906)⁽²⁾, Dawson(1926)⁽³⁾, Lipman (1935)⁽⁴⁾, Cumley(1939)⁽⁵⁾, True(1947)⁽⁶⁾, Dingerkus(1977)⁽⁷⁾ and Potthoff(1984)⁽¹⁾.

Inouye reported Alizarin red – alcian blue double staining on skeletal and cartilage tissue⁽⁸⁾. Alizarin red – Alcian blue double staining is a simple yet an effective and inexpensive method for observing and evaluating the appearance and small details of fetus or skeletal system without using expensive instruments like an x-ray⁽⁹⁾. Structural appearance evaluation using this technique allows for better understanding of the performance of anatomical system and evaluation of body structure of different species⁽¹⁰⁾. Clearing and staining of bone with Alizarin red is an important method to study the skeletal differences and development of vertebrates. Sometime later Taylor used trypsin to digest the proteins in the specimen allowing it to become transparent in glycerol to clearly visualize the skeleton.

The aim of the present study was to present the experience and improvisation protocol for staining an Ovariectomised and non-ovariectomised rat to note the skeletal and cartilage abnormalities. The purpose of the study was to apply the diaphonization technique for preserving the samples dyed by Alizarin red – Alcian blue double staining method and preserving the specimen in glycerin filled containers. Finally the colour quality , tissue transparency and other form of skeletal deformities of the samples were compared with each other.

MATERIALS AND METHODS:

Chemicals:

The chemicals required for the staining process are Given in Table – 1.

STEPS	CHEMICALS REQUIRED
Fixation	10% Formalin and Distilled water
Washing	Distilled water
Cartilage staining	Alcian blue , 95% Ethanol , Glacial acetic acid
Rehydration	95% Ethanol , Distilled water
Trypsin digestion	Sodium borate , Distilled water , Trypsin
Bone staining	Potassium hydroxide , Alizarin red S , Distilled water
Clearing	Potassium hydroxide , Glycerin
Storage	Glycerin, Thymol crystals

EXPERIMENTAL OVERVIEW:

Step 1 - Prepare, Fix and Wash

Remove

Step 2 - Cartilage Staining

Step 3 – Rehydration

Step 4 – Trypsin digestion

Step 5 – Bone Staining

Step 6 – Clearing, Bleaching and Storing

Three month old Female Wistar rats^(22,23) were used for the study (n = 2). One animal was used as control and the other animal was ovariectomised. Bilateral Ovariectomy was performed using cocktail anesthesia of Ketamine and Xylazine (10mg/kg). Ovariectomy was done by making a small incision of 1 cm in the dorsal side. The ovary surrounded by fat was identified and the blood vessels supplying to the ovary was ligated and the ovary was cut off. The wound was closed using absorbable sutures. Povidone iodine solution was sprayed on the wound. Immediately after surgery tramadol (5mg/kg) was given intra-peritoneal to reduce the post-operative pain⁽¹¹⁾⁽¹²⁾. The animal after surgery was kept in an individual cage for a week for the development of osteoporosis.

The following steps were followed individually for each specimen preparation

PART 1 – Preparing the specimen

The animals were euthanized with excess of thiopental sodium (50mg/kg). The animal’s skin was removed followed by the organs and the surrounding fat. The skinning process was done to avoid the interference of the tissues with the clearing process. The specimen was then thawed carefully and completely⁽¹³⁾. Once the skin was completely removed the abdomen was cut open to remove all the organs including the heart and the lungs. Finally the fat was removed and the specimen was kept in 10% formalin solution.

PART 2 – Fixation

Once the fat and the organs were removed the animal was fixed in formalin. A 10% formalin solution was prepared and was poured into the specimen bottle. It was carefully done and made sure that the specimen was completely covered with the formalin solution [Fig 1]. The specimen was left in the solution without disturbing for 2 – 3 day^s⁽⁹⁾.

PART 3 – Washing

After fixing the specimen in formalin for few days it was washed in order to rehydrate the tissues to remove the excess formaldehyde. The specimen went through a series of water washings as follows

Day 1 – fill it with distilled water and leave it overnight

Day 2 – fill it with distilled water and leave it overnight again

Day 3 – transfer the specimen to the formalin solution and leave it for some 1 – 4 hours, then remove it and fill it with distilled water

Day 4 – fill it with fresh distilled water and leave it overnight

Day 5 – repeat the procedure which was done on day 3 and day 4

Day 6 – the washing procedure is repeated until the bones are exposed

The distilled water washings was done till the bones were clearly visible. Once the bones are visible the cartilage staining was done⁽⁸⁾ [ Fig 2].

PART 4 – Cartilage staining

Once the washings were done properly the cartilage has to be stained. The required quantity of alcian blue was weighed. In an empty jar the alcian blue powder was added followed by 95% of ethanol followed by addition of glacial acetic acid and was mixed thoroughly. The specimen was transferred to the jar containing the dye. It was made sure that the specimen is completely immersed in the dye solution. The specimen was soaked in the dye for one day before moving it to the rehydration process⁽¹⁴⁾[ Fig 3].

PART 5 – Rehydration

The specimen after the cartilage staining went through a series of rehydration process. For the rehydration process the specimen was given a series of ethanol washes. Various percentage of ethanol was prepared and kept for rehydration.

Bath 1 – 95% ethanol

Bath 2 – 95% ethanol

Bath 3 – 70% ethanol

Bath 4 – distilled water

Day 1 – Bath 1

The specimens were transferred to the jar containing 95% ethanol until it was covered completely and made to sit overnight.

Day 2 – Bath 2

Same procedure which was done on day 1 was followed and the was allowed to sit

Day 3 – Bath 3

70% of the ethanol solution was made and the specimens were transferred to the solution and allowed to soak for one day.

Day 4 – Bath 4

The ethanol was completely drained and the specimen was given a few washings with distilled water and then finally it was refilled with fresh distilled water and allowed to soak for a day.

PART 6 – Trypsin digestion

Trypsin is a digestive enzyme which breaks down the protein leaving the collagen. This breaking down of the muscles and other tissues will help the specimen become transparent while the collagen will prevent the specimen from falling apart. The trypsin digestion was done to remove the adhering tissues in the specimen. Trypsin works better when it is at an p H of 7.5 – 8.5. To make the trypsin solution boil the distilled water and add the weighed quantity of the borax powder and mix it well until it gets dissolved completely. Allow the borax solution to cool down and add the trypsin powder. Pour the ingredients into a well cleaned empty jar and transfer the specimen into the solution until it is completely immersed. The specimen was checked regularly for its digestion process. The solution was changed every 3 days until the tissues wither off. Once the specimen becomes completely visible it was best to proceed with the bone staining process⁽⁷⁾.

PART 7 – Bone staining

Once the trypsin digestion was done the next step was to stain the bones. Required quantity of potassium hydroxide was weighed and added to an empty glass container followed by distilled water. The sufficient quantity of Alizarin red S powder was added to the solution of potassium hydroxide and the specimen was completely immersed in it. The specimen was kept in the dye solution for 1 day⁽¹⁵⁾ [Fig . 4].

PART 8 – Clearing

The best and longest part of the staining process. The longer the time the specimen is being cleared the better the visibility of the bones and the cartilage. For the clearing process the specimen went through a series of clearing process.

Bath 1 – 0.5% KOH in 50 ml of glycerin

Bath 2 – 0.5% KOH in 75 ml of glycerin

Bath 3 – 0.5% KOH in 150 ml of glycerin

Transfer the specimen to bath 1 of the KOH and glycerin solution and incubate it for a day. Similarly the procedure is done with bath 2 and 3 until the specimen is free from any adhering tissues⁽³⁾.

PART 9 – Results

Once the clearing process is done the specimen has to be fixed permanently in glycerin. The specimen was taken out and any adhering loose tissues and the other imperfections were removed. The specimen was then kept in a clean glass jar and it was filled was glycerin until it was completely immersed. Finally few crystals of thymol was added as an antifungal agent [ Fig 5].

RESULTS:

Bones and cartilages of the skeletal parts of the stained sample turned into red and blue respectively indicating the presence of bone and cartilage. The samples were so clear that the skeletal parts were clearly seen. The absorbed colors were stable and did not fade away in course of time. At the end of the staining process the other tissues and muscles did not absorb any color. Samples kept their rigidity throughout the staining process. The control animal (non-ovariectomized) showed good staining of the bones and the cartilage. While the ovariectomized animal showed less staining of the bones and no staining of the cartilage.

Fig. 1

Fig .2

Fig .3

Fig.4

Fig.5

DISCUSSION:

Diaphonization is the process of clearing and staining the specimen developed by G.Dingerkus and L.D.Uhler was an alternative means of studying anatomy on smaller animals. It is a process by which the chemicals are employed to render the skin of an animal translucent as well as break down the flesh while the dyes are employed to color the bones and the cartilage. A complete translucent specimen is not usually interesting or useful, so in order to complete the diaphonization process we turned to the use of dyes. The specimens are dyed with either alizarin red or alcian blue.

Alizarin red S^(19)(20) is used in histopathology mainly to stain or locate calcium deposits in tissues. In the presence of calcium alizarin red s will bind to the calcium to form pigments that is orange to red in color. Whole specimens can be stained with alizarin red s to show the distribution of bone. Alcian blue is a byproduct of acid making process. The only thing the dyes have in common is their affinity for calcium where they encounter it and stick to it. This is because the bones are filled with calcium they uptake the dye and stand out red or blue in the translucent remains of the flesh.

The specimen showed red staining of the bones and blue staining of the cartilage so that the anomalities of both the cartilaginous and the bony skeleton were examined carefully⁽²²⁾. This method provided good visualization of the bone surface as well as profiles allowing the detection of irregularities of a developed bone. The excellent visualization the skeleton was made possible by the use of trypsin⁽²¹⁾which cleared the surrounding soft tissue and heavy pigmented deposition. The degree of transparency of soft tissues was attained based on the trypsin digestion and the amount of formalin used. Since the specimen was fixed in formalin it showed good staining of both the bone and cartilage and it was easier to handle during the skinning and the evisceration process.

This double staining of the specimen came out with excellent results. The OVX animal was compared with the control animal. The OVX animals showed less staining when compared with the control animal. The bones were colored in red but not as that brightly when compared with the control animal. The cartilage was completely absent in the OVX animal. The absence of cartilage might be due to decreased calcium deposition in the bones after the Ovariectomy procedure. The control animal had sufficient amount of calcium due to which it got stained with alcian blue and alizarin red S showing the presence of calcium deposition in the cartilage⁽²⁴⁾ as well as bones.

Finally it can be reported that estrogen deficiency⁽¹⁷⁾ might lead to less deposition or nearly no deposition of calcium in the bones⁽²³⁾ and cartilage. Previous studies have reported decreased calcium in Ovariectomized rats⁽¹⁶⁾.Calcium is one of the most important mineral in the formation of bones. Having low levels of calcium content might be a major risk for the formation of brittle bones and muscle weakness. Proper maintenance of calcium is not only important for bone growth⁽¹⁸⁾ but also to protect the bone strength. Estrogen supports this activity by aiding in intestinal calcium absorption. Having low estrogen levels negatively impacts the body’s ability to make use of calcium. In course of time with this less or decreased deposition of the calcium the skeletal system tend to loss its strength and integrity leading to brittle nature. So it can be concluded that estrogen deficiency might lead to decreased absorption.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of SRM College of Pharmacy, SRM IST , Kattankulatur for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 17.09.2019 Modified on 01.11.2019

Research J. Pharm. and Tech 2020; 13(5):2228-2232.

DOI: 10.5958/0974-360X.2020.00400.X