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JOURNAL OF
THE ANATOMICAL SOCIETY OF INDIA

Vol. 49, No. 2, December, 2000


In this issue :

Editorial
Dr. Patnaik V.V.Gopichand

Gross Anatomy of the Caudate Lobe of the Liver
Sahni, D., Jit, I., Sodhi L. Department of Anatomy, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Branching Pattern of Axillary Artery - A Morphological Study
*Patnaik V.V.G., Kalsey, G; Singla Rajan, K. Department of Anatomy, Government Medical College, Amritsar, *Patiala. INDIA

The Course, Relations and The Branching Pattern Of The Middle Meningeal Artery In South Indians
Manjunath, K.Y. & Thomas, I.M. Department of Anatomy, St. John�fs Medical College, Bangalore-560 034 INDIA

Morphometry of the Human Inferior Olivary Nucleus
Dhall, U; Chhabra, S. & Rathi, S.K. Department of Anatomy, Pt. B.D. Sharma P.G.I.M.S., Rohtak. INDIA

Management of Turner Syndrome in India Using Anthropometric Assessment of Response to Hormone Replacement Therapy.
Sehgal R. and Singh A. Department of Anatomy, Maulana Azad Medical College and Associated Lok Nayak, G.B. Pant & G.N.E.C. Hospitals, New Delhi ? 110 002 INDIA.

Insertion Of Umbilical Cord On The Placenta In Hypertensive Mother
Rath* G, Garg** K, and Sood*** M. *Department of Anatomy, ***Department of Obstetrics & Gynaecology, Lady Hardinge Medical College, New Delhi-110001 **Department of Anatomy, Santosh Medical College, Gaziabad. INDIA

Utility Of Finger Prints in Myocardial Infarction Patients
Dhall, U; Rathee, S.K; *Dhall, A; Department of Anatomy & *Medicine, Pt. B.D. Sharma, PGIMS, Rohtak. INDIA

The Prenatal Parotid Gland
Fouzia Nayeem, Sagaff S., *Krishna G., **Rao S. Department of Anatomy, K.A.A.U. Jeddah. Department of *Pediatrics & **Surgery, Osmania Medical College, Hyderabad. INDIA

Possibility of Cell Death Induced Skeletal Malformations Of The Upper Limb
Sinha, D.N. Department of Anatomy, B.R.D. Medical College, Gorakhpur?273013 INDIA,

Efficacy of Manual Bladder Expression in Relieving Urine Retention After Traumatic Paraplegia In Experimental Animals.
Preeths, T.S., Sankar, V. Muthusamy, R. Department of Anatomy, Dr. A. Lakshmanasamy Mudaliar Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India.

Stress And Serum Cholesterol Levels-An Experimental Study
Jain, S.K. *Pandey, S.N. *Srivastava, R.K. Ghosh, S.K. Department of Anatomy, D.R.P.G. Medical College, Kangra at Tanda. * Department of Anatomy, G.S.V. Medical College, Kanpur.

Effect of Ibuprofen On White Cell Series of Bone Marrow Of Albino Rats
* Bhargava, R., Chandra, N., Naresh, M., *Sakhuja S. * Department of Anatomy, M.L.N. Medical College, Allahabad * Lady Hardinge Medical College, N. Delhi, India.

JB4 An Embedding Medium For Flourescent Tracer Technique
*Gupta, M; **Mishra, S., ***Sengupta P. Department of Anatomy, *PGI, Chandigarh; **AIIMS, N. Delhi; ***UCMS, New Delhi. INDIA

Comparative Anatomy of Cardiac Veins in Mammals
Kumar Keshaw Department of Anatomy, Institute of Medical Sciences B.H.U., Varanasi?5. INDIA

Aplasia Cutis Type 9 With Trisomy-13 Syndrome ? A Rare Association
Adhisivam, B, Narayanan, P, Vishnu Bhat, B, *Ramachandra Rao. R*, *Rao. S*, Kusre, G.* Department Pediatrics & *Anatomy, JIPMER, Pondicherry - 605 006

Absence of Musculocutaneous Nerve And The Innervation of Coracobrachialis, Biceps Brachii And Brachialis From The Median Nerve
Sud, M.; Sharma A. Department of Anatomy, Christian Medical College, Ludhiana. Punjab INDIA.

A Rare Pseudo Ansa Cervicalis: A Case Report
Indrasingh I. and Vettivel S. Department of Anatomy, Christian Medical College, Vellore, India

A Rare Variation In The Relation Of Omohyoid Muscle: A Case Report
Vettivel, S. Korula, A. and Koshy S. Department of Anatomy, Christian Medical College, Vellore, India

Surgical Incisions ? Their Anatomical Basis Part II - Upper Limb
1Patnaik V.V.G., 2Singla Rajan. K., 3 Gupta P.N. Department of Anatomy, Government Medical College, Patiala1, Amritsar2, 3Department of Orthopedics, Government Medical College, Chandigarh. INDIA

Anatomy Of Temporomandibular Joint?A Review
1Patnaik V.V.G., 3Bala Sanju; 2Singla Rajan K. Department of Anatomy, Govt. Medical College, 1Patiala, 2Amritsar, 3Department of Oral & Maxillofacial Surgery, Pb. Govt. Dental College, Amritsar


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J Anat. Soc. India 49(2) 169-171 (2000)
JB4 An Embedding Medium For Flourescent Tracer Technique

*Gupta, M; **Mishra, S., ***Sengupta P. Department of Anatomy, *PGI, Chandigarh; **AIIMS, N. Delhi; ***UCMS, New Delhi. INDIA

Abstract : Frozen sections are ideal for studying fluorescence tract tracer technique. In the present study instead of using frozen sections, JB4 embedding plastic sections have been used to study the flourescence of fast blue, a fluorescent tract tracer. Fast blue was instilled into the right sciatic nerve in 20 albino rats weighing between 150-200 gms of body weight. On the 5th day the dorsal root ganglia and spinal segment of L3-SI were removed. The tissues were processed and embedded in JB4, a water soluble embedding medium. Ventral horn cells of right L4, L5 and L6 segments showed the evidence of fluorescence. Every 5th section was stained with thionine for light microscopy. The advantage of these resin sections were that the technique was simpler, easier and faster with minimal shrinkage.

Keywords : JB4, Flourescence, Embedding medium.

Introduction :

Polymerising resins were introduced in the form of methyl methacrylate (Pease 1964). Later water soluble ester glycol methacrylate (GMA) came into use (Bennet et al 1976). The sections can be prepared by an ordinary microtome at room temperature. The water soluble property of glycol methacrylate obviates the need for dehydration (Bennet et al 1976). It can be used for routine histopathological and experimental studies for light microscopy as well as fluorescent microscopy. It has been successfully used for enzyme histochemistry (Higuchi et al 1979, Hoffman and Teresa 1981, Cole 1982, Murray and Ewen 1990) and immuno-histochemistry (Casey et al 1988).

JB4 embedding kit, used in the present study is made by Polyscience lab, Warrington, Germany. It contains glycol methacrylate as the embedding solution.

JB4 kit has three components:
Solution �gA�h ? glycolmethacrylate
Solution �gB�h ? N-N dimethyl aniline
Dry catalyst ? Organic peroxide

Material and Methods :

The present study was conducted in 20 adult albino rats weighing 150-200 gms of body weight. The animals were anaesthetised with anaesthetic ether. The right sciatic nerve was exposed distal to the ischial tuberosity under aseptic conditions. A polythene pouch containing 1m litre of 2% aqueous solution of fast blue was tied around the severed proximal end of the nerve. The animals were observed post-operatively and kept in plastic cages. They were given food and water ad libitum.

On the 5th day the animals were sacrificed and perfused with 10% formalin. The sciatic nerve was dissected out from the spinal cord along with the corresponding dorsal root ganglia (L3-SI). The dissected spinal segments and ganglia were kept in 10% sucrose cacodylate buffer at pH 7.2, 2�‹C for 24 hours, till the tissue sank to the bottom. This was followed by JB4 processing.

Processing by JB4.

A stock solution of solution �gA�h was prepared by taking 100ml of solution �gA�h and dissolving 1gm of dry catalyst into it. The prepared stock solution was stored in the refrigerator at 2�‹C. The tissue was removed from the buffer and a daily change of stock solution �gA�h was given for three days at 2�‹C. The tissue became hard between hard and translucent and ready for embedding.

Embedding

To 1u litre of solution �gB�h 25m litre of solution �gA�h was stirred vigorously on an ice bath. In a plastic beam capsule the embedding fluid was added and then the tissue was kept vertically. The capsule was kept in a stand. The anatomical orientation was maintained by inserting a piece of paper on the right side of the tissue. Slowly the embedding fluid was poured into the capsule and the lid was tightly secured so that no air could enter it, as air causes premature polymerisation. It was kept for 12-24 hours till the blocks were hard. The blocks were removed by cutting the capsule by a razor blade.

Section cutting

The blocks were trimmed with a sharp blade and 20m thick sections were made on a rotary microtome using a steel knife. The sections were picked up by a thick brush and arranged on a clean slide. The sections were made to expand and were prevented from curling up by adding a drop of ammoniated water on them. The sections were air dried and observed under a Zeiss fluorescent microscope using an excitation filter of 365 nm and a barrier filter of 397 nm. The fluorescence in the ventral horn cells and dorsal root ganglia of the right side was compared with that of the left side which served as control.

Every 5th section was stained with Cresyl violet to study the morphology.

Observations :

The ventral horn cells of L4, L5 and L6 segments of the spinal cord showed evidence of fluorescence. The fluorescence was intense in the cytoplasm and also in the cell processes. There was a distinct negative nuclear shadow (Fig 1). The pseudounipolar cells of the dorsal root ganglia of L4, L5 and L6 segments also showed evidence of fluorescence on the right side (Fig 2). The sections stained with cresyl violet showed distinct Nissl substance in the cytoplasm with euchromatic nucleus (Fig 3). The cytoarchi-tecture was very clear.

Discussion :

Fast blue fluorescent tracer is taken by retrograde axonal transport. Its uptake is similar to HRP (Steward 1981). It preferentially labels the cytoplasm of cell bodies and dendrites and gives a negative nuclear shadow (Bentivoglio et al 1980). In the present study fast blue was taken by sciatic nerve neurites at the site of instillation and L4, L5 and L6 spinal segments and dorsal root ganglia showed the presence of intense fast blue fluorescence with a negative nuclear shadow. Valda de Valda (1980) found plastic embedding to be simpler and easier than frozen sections for fluorescent studies.

Samba (1979) used semi thin sections by the conventional rotary microtome by using a Ralph glass knife. In the present study it was possible to make 5m thick sections by glass knives made from micro slides. Since we required 20m thick sections a steel knife was preferred. The main disadvantage of JB4 embedding, which we faced, was softening of the blocks. However, increasing the concentration of the dry catalyst could harden the blocks. Samba (1979) reported that dry heat improves the hardness of the block. This couldn�ft be used as heat causes fading of fluorescence. Adding chemicals like Divinyl benzene and methyl methacrylate monomer to the embedding solution also increases the hardness (Samba, 1979). Another disadvantage of JB4 was that we could not get a ribbon by JB4. Eric, (1972) devised a procedure in which he trimmed the cutting surface of the block of the tissue into a rectangular surface. The block was coated on all sides with contact cement which could float in water.

The advantage of JB4 embedding medium is that a small tissue like dorsal ganglion can be processed and cut into sections easily. Mohammed et al (1988) used this medium for processing the dorsal root ganglion. They cause less distortion and provide better results on staining (Bennet et al 1976) This was also observed in the present study. The processing is quicker and easier. Sections are prepared at room temperature by an ordinary rotary microtome. The water-soluble property of glycolmethacrylate causes no need for dehydration. The blocks are easily made and less time consuming. Hence this is an ideal medium for small sized tissues.

References

1. Bennet, H. S., Wyrick, D., Lee, S.W., McNeill, J.H. (1976): Science and art in preparing tissue embeded in plastic for light microscopy with special reference to glycol methacrylates, glass knives and special stains. Stain Technology; 51(2): 71-79.
2. Bentivoglio, H.G.J.M., Kupeyrs, C.E., Berrevoiets, H.L. and Dann, O.(1980): The two new fluorescent retrograde neuronal tracers which were transported over long distances. Neuroscience letters; 18: 25-30.
3. Casey, T.T., Causer, J.B. and Collins, R.D. (1988): A simplified plastic embedding and immunohistological technique for immunophenotypic analysis of human haemopoetic and lymphoid tissues. American Journal of Pathology; 131(2) : 183-184.
4. Cole, M.B. (1982): Glycol methacrylate embedding bone and cartilage for light microscopic staining. Journal of microscopy; 127(2) : 139-148.
5. Eric, C.H.D. (1972): A method for obtaining ribbons of serial sections of plastic embedded specimens, Stain Technology; 52: 59-60.
6. Higuchi, S., Moritaka, S., Dannenberg, A. M. and Brian, H; (1979); Histochemical demonstration of enzyme activities in plastic and paraffin embedded tissue sections. Stain Technology; 54(1): 5-11.
7. Hoffman, E.O., Teresa, R.F. (1981): High resolution light microscopy in renal pathology. American Journal of Clinical Pathology; 76(5): 636-643.
8. Mohammad, A. A., Parker, T.L. and Coupland, R.E. (1988): The innovation of the adrenal gland II. The source of spinal afferent nerve fibres to the guinea pig adrenal gland. Journal of Anatomy; 160: 51-58.
9. Murray, G. I. and Ewen, S.W.B. (1990). Enzyme histochemistry on freeze substituted glycol methacrylate embedded tissue. The Journal of Histochemistry and Cytochemistry; 28(1): 95-101.
10. Paese : Histological techniques for electron microscopy In : �e�eEmbedding 2nd edn. Academic Press New York. 11: 82-135. (1964)
11. Samba, R. (1979). Contribution to semithin sectioning on a conventional rotary microtome. Stain Technology; 54(5): 251-255.
12. Steward, O, Neuronatomical tract tracing method In : Horse radish peroxide and fluorescent substances and their combinations with other techniques. L. Heimer and M.J. Rebards. (eds). Plexum Press, New York: 279-310. (1981)
13. Valda de Valda, S.H.T. (1980). Fluorescent microscopy of glycol methacrylate embedded tissue. American Journal of Clinical Pathology; 73(1): 119-121.


Fig. 1. Photmicrograph of JB4 embedded sections of the rat spinal cord at L5, level, showing Fast blue fluorescing ventral horn neurons. The cytoplasm is fluorescing while the nucleus shows a distinct negative nuclear shadow. X 400


Fig.2. Photomicrograph of JB4 embedded sections of the rat dorsal root ganglion at L5 spina segment level. Fast blue fluorescene is present in the pseudounipolar cells of the dorsal root ganglion.


Fig. 3. Photomicrograph of JB4 embedded sections of the rat spinal cord at L5 level, showing the ventral horn neurons stained with creasyl violet. Large neurones with distinct cell processes are visible.



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