Plan for June!

This is the Plan for this month.

• Thesis completion - pathology department - morphometric analysis of mice spermatogenesis
• thesis - BK - photoshop the photographs taken especially Xrays
• thesis MJ - writing the basic introduction and review of literature, with bibliography
• Prepare for the State conference- award paper - 
• Prepare for paper - comparative histology of tongue
• Prepare a poster and paper for SK and Sai
• Read for the upcoming Neuroanatomy test
• Blog 
• Write the article - morphometric analysis of Stapes
• Look into morphometry of lumbar vertebra
• Look into PCOD ovary anatomy

Histomorphometry of the slides still not done

Though most of the work is over, the final morphometric analysis of the slides prepared from the mice testis is still pending.
For this assistance of the department of pathology is sought. They too are willing. I must accommodate some time at least next week for this.
The following parameters are intended to be measured in the slides prepared so far, using a morphometric microscope.
10 semeniferous tubules which are cut almost transversely in each slide is taken for the account. Three such observations will be done from each of the testis from non consecutively cut sections.

1. Semeniferous tubular diameter, surface area and perimeter
2. Maximum epithelial cell height from the tubular basement membrane
3. Sertoli cell nuclear diameter
4. Myoid cell count
5. Minimum and maximum interstitial space length 

From the sperm smear slides the following are intended to be noted.

1. Sperm head shape variations.
2. Decapitated sperm heads per 1000 sperms counted
3. Vacuolation of sperm head per 1000 sperms counted.

Mean while we had a MCI inspection in our college, to which my honorable guide has attended and he generously signed the declaration page to be pinned with my thesis copy later on. 

Write up of thesis

Though the thesis is not yet complete, I have started the write-up work. Only probable remaining work in the thesis is sacrificing, dissection and histology of so called group D mice, wherein it is expected to see a reversal of drug effects.
It may be followed by sacrifice of offsprings of mice treated with drug; if there are any changes, its a delight.

these are the changes seen in histology.

testis of control mice.

testis of mice treated with 4 mg of gemcitabine.

changes identified so far
1. decrease in seminiferous tubule morphology - decrease in its diameter. quantification required and significance has to be established.
2. change in the cytoarchitecture of seminiferous tubules
3. Interstitital tissue has decreased. But tightly packed with cells
4. large nucleoli seen in spermatogonial cells
5. Vacuoles seen in few spermatogia.
6. Probable increase in sertoli cell
7. Probable increase in Leydig cells.
8. Decrease in spermatids.
9. Uniform hyalinized appearance of the lumen of tubules.
10. thickening of basement membrane of tubules.
11. decrease in height of epithelial cells.

these have to quantified.

Sperm shape abnormalities:

apparently normal sperms

mice treated with gemcitabine

observations so far -

1. many of the heads are decapitated.

2. loss of banana tailing.

3. vacuolation of the cytoplasm in the head.

these things again has to quantified and significance has to be proved with reference to normal.

2nd round of mice sacrifice!

Today 7 mice were sacrificed with a rat as per schedule.

The tissues from the mice – testis and ovary are collect in Bouin’s solution and allowed to get fixed.

The tissues which were collected last week, which waited till today, getting ‘fixed’ was given to Department of pathology for further processing, paraffin embedding, sectioning and staining. Though they were unhappy about the extra work they got because of our tissues, I am relieved as I don’t have to struggle in my department putting up the lab. More over I don’t have to slog with our assistant professors for the preparation of histological slides.

Out of 7 mice dissected one female mice was concieved and it had 5 intrauterine concepts.

Some of the images taken during dissection are below.

Abdominal exposure of the mice, intestines are out. Working in the area of kidney and uterus in an attempt to isolate the ovary.

This is me and BK sir. We dissected mice in our dissection hall - in the gap between the practical and viva voce for the 1st year MBBS students, 1st sessional examinations are on!

Pregnant uterus dissected intoto with the ovaries and Fallopian tubes

4 Mice died! But Experiment is ON.

It was all fine for the first two days. On the third day I found a female mice dead from the 4 mg group. I took the corpus and dissected it in our department with SK sir. But by the afternoon, it was badly smelling and we could not bear it till we could possibly find its ovaries! And I have no idea where its ovaries were! I got some fat from the omentum about the calculated size of gonad and assumed it as ovary!!

The next day – while taking dissection I got a call – Akka, informed me that another mouse is dead. It was again a female, from the group which received 4mg drug. I discarded the corpus straight away!

The next day – Sunday – after the morning ‘tulu mass session at Milagris’ we entered the animal house taking the name of the god and found a male (8mg group) and a female (4 mg group) dead. I know it is unscientific in any study to discard the corpus without knowing the cause of death.

Since then there are no more casualties.

Today I and BK sacrificed 5 mice (3 male+ 2 female) by simple cervical dislocation. We could identify the ovaries and pancreas which we had difficulties in earlier attempts. I have taken the tissues – testis and ovaries in Bouin’s solution and pancreas and kidney in regular 10% formalin.

The story so far-  all mice were injected with Gemcitabine on 23-11-10.

No. of mice injected with drug	Approximate amount	Mice dead	Date of death	Number of mice sacrificed to be on
				01-12-10	07-11-10	30-01-11
6 male mice	4 mg each			2	2	2
4 male mice	8 mg each	1	28-11-10	1	2
5 female mice	4 mg each	1 1 1	26-11-10 27-11-10 28-11-10	1	1(-1)	0
4 female mice	8 mg each			1	2	1(+1)

We are left with 19-4-5=10 mice.

Next Tuesday – 7 mice are scheduled to die.

I am left with 3 to test the reversibility of the drug later in January.

The histological processing of the tissues have started, we are running a trial, by using human liver and rat spleen. Tomorrow it will take the proper shape. By then I will process the mice tissues and get ready for the sectioning and staining by Thursday.

Will post the photos of mice tomorrow.

Experiment started!

Today we injected the drug!

Some how the department of Pharmacology is persuaded and they agreed to give the aged mice - which they are not using. Yesterday Prof and Head of Pharmacology directed Akka to give the mice. I received 10 male and 9 female Swiss albino mice in two cages. I made the necessary purchasing of diluent for Inj. Gemita - that's NS, with 20 insulin syringes.
Today - with BK sir and Santhosh sir we injected the mice with Gemcitabine and divided them into 2 groups - those which got 4mg and those which got 8mg!

No. of mice injected with drug	Date	Approximate amount	Number of mice sacrificed to be on
			30-11-10	07-11-10	30-01-11
6 male mice	23-11-10	4 mg each	2	2	2
4 male mice	23-11-10	8 mg each	2	2
5 female mice	23-11-10	4 mg each	2	2	1
4 female mice	23-11-10	8 mg each	2	2

Totally 19 mice are subjected to the study and we have diluted the entire drug in the ampule !!
We are left with more than half of the drug - most probably will be discarded tomorrow !

Tomorrow will be posting the photos !

Mice procurement - not so easy!

The department of Pharmacology has established the central animal house and in charge of it. It has taken a heavy toll on the personnel involved in the work, and are in colossal hope of the rewards for their solid work.

From our side – we always are in tough fight against materialistic attitude of the people around us, making fun of people, are using the same material which they are bargaining for their survival! It’s a tough call, but every time an egoistic face will unearth and will wipe the whole idea of collaboration and corporation.

So in these conflicts the idea of whose animal was it, which invariably not going to make up for the race of survival but eventually going to die – will stand as a mockery of all the human efforts on the surface of earth and eruct the walls of hatred for materialistic, mortal men and at the same time, make us feel the eternity of the concept that we are earthly!

Of late we came to know that the people in pharmacology didn’t like the concept of using the animals by us, which they have purchased for money.

I have made a clear notion- that the animals for our experiment will be purchased just as they are doing. Probably that’s the only way of making it.

I also got the clear notification that BK can get away with it. And any irregularity on the part of animal procurement, handling and sacrificing will have a serious face and consequences if unforeseen mishappening occur with me.