Cercetări privind indicii morfo-fiziologici şi ... · Cercetări privind indicii morfo-fiziologici şi modificările rinichiului în diabetul experimental la şobolanii din linia

Cercetări privind indicii morfo-fiziologici şi modificările rinichiului în diabetul experimental la şobolanii din linia Wistar

Gabriela Zoiţa Zah 25

SUMMARY

Our research had as main aim of knowledge he suffered kidney changes

caused by subclinical diabetes and streptozotocin treatment outcome original herbal products.

Diabetes is a complex disease with clinical expression varied in form and intensity, in appreciation of his research is needed clinical, biochemical, physiological and metabolic complex and involve the relational interpretation of all the processes that accompany it in the end to address a therapy and prophylaxis especially good to succeed. For that I followed:

- Changes in blood biochemical parameters of the main disease and treated animals;

- Changes found in animal hematology disease and treated with herbal products usual antidiabetic medication compared to the original;

- Kidney histopathological changes found in the experiment; - We intend to do a study and bibliography of the biochemical role of minerals

involved in elasticity and permeability of capillary vessels; The purpose of this paper is to correlate the clinical aspects of biochemical,

hematological and histopathological conditions which meet and ultimately are responsible for diabetes.

Because diabetes itself is a disease with a long evolution, the observations we have made a 7-month illness and treated animals.

Objective in addressing this issue and making this work: - Updating data on subclinical diabetes in humans and animals; - Reproduction of subclinical experimental diabetes in rats, male Wistar race; - Development and monitoring of animals for their accommodation; - Provide standard conditions of hygiene, food and microclimate; - Follow daily treatment made animals for 7 months; - Periodic collection of blood samples, their processing of primary and specific

tests done; - Sampling of kidneys for histological analysis; - Full respect for ethical measures regarding the protection of animals

subjected to experiments; - Data processing, interpretation, compliance graphics; - Statistical processing of data which lend themselves to greater safety

significance; - Specific processing of histological samples, interpretation of results and

illustrate their photo images. MATERIALS AND METHODS

The research was done on 63 male rats race ends Wistar two months old, elected as class (mass 150 g), age and size to be as uniform.



At first I took randomized seven heads, and we formed the first batch: - Lot 1 healthy - 7 layers - neîmbolnăvit, healthy controls.

Other animals were injected with 4 mg / 100 g body weight ip Streptozotocin The animals were monitored three days, then we form experimental groups as follows: Lot 2 consists of 14 animals - sick and untreated control group. Lot 3 consists of 14 animals - group treated daily with 1 tb SIOFOR 1000, equivalent to 12 mg / kg and 4 g lactose to correct taste. Lot 4 consisting of 14 animals - test group by 5.4 g daily ERIDIAROM / lot / day. Lot 5 consists of 14 animals treated daily with DIAVIT, 6 g / lot / day.

Housing, feeding and watering the animals was the same (food standard) also microclimate conditions.

The investigations were made during 2007 - 2008 on a herd of 63 heads rats. After the first month, the 2nd, 3rd, 5th and 7th month experimental blood

samples were collected (from every 7-8 animals / group) for serological and biochemical tests. Individual samples were taken from internal angle of the eye, were collected in containers specially trained, kept on ice and transported to a time when biochemical research laboratory.

After the first month, the 2nd, 3rd month, the 5th and 7th were killed by two animals (groups 2-5) for histopathological examination and 2 animals in group I compared to healthy controls (7 months). Samples were fixed for histopathological examination performance.

Mention that during the experiment actually came out of (dead) the following animals: - 1 head of lot 1 - healthy control group;

- 2 heads of lot 2 - sick untreated control group; - One head of group 4 - treated with Eridiarom and lot 5 - treated Diavit

Animals were followed clinically from throughout the experiment. Histological processing of samples consisted of fixation in 20%

formaldehyde, then shape each sample, including paraffin, microtome and then cutting the fixation and staining with Tricromic (HE and blue), then the optical microscope examination and photography images are representative.

Biochemical and hematological examinations were processed individually by specific laboratory methods.

Biochemical examination were followed: serum calcium and total magnesium, glucose, serum urea and creatinine.

Haematological examination were pursued: leukocytes, lymphocytes, hemoglobin, hematocrit and platelets.



RESEARCH AND RESULTS BIOCHEMICAL INDEXES TRACKING RATS FOR 7 MONTHS

Total serum calcium

Table 1 Average calcium values in the study groups (mg/dl)

Lun

a /

mon

th

Nr.

Indi

v. /

no. I

ndiv

. Valori normale/ Normal

value

Lot martor sănătos / Healthy

control group

Lot bolnav netratat / Untreated

sicked group

Lot bolnav tratat cu Siofor / Group

treated with Siofor

Lot tratat cu Eridiarom /

Group treated with Eridiarom

Lot tratat cu Diavit / Group

treated with Diavit

1 6 4.5 – 5.5 4.42 4.88 5.18 5.12 4.76

2 5 4.5 – 5.5 4.17 5.38 5.26 5.4 5.3

3 7 9 – 9.5 7.99 5.36 9.33 9.60

5 6 9 – 9.5 8.725 8.083 9.38 9.56

7 6 9 – 9.5 8.068 7.974 9.18 9.389

After 2 months of treatment there is a significant increase in serum calcium

most important in the group treated with Eridiarom followed by untreated patient group (10.76), then the group treated with SIOFOR and group treated with Diavit.

Between 5-7 months of observation, the group of calcium increased slightly ill but does not reach the lower limit of normal while the groups treated with Eridiarom and Diavit values fall within the upper limit of normal.

33.5

44.5

55.5

66.5

77.5

88.5

99.510

1 luna 2 luni 3 luni 5 luni 7 luni

SanatosHealthyBolnav Sick

Diavit

Eridiarom



Graphic 1 Calcium evolution 1 – 7 months

GLUCOSE (GOD/POD) Table 2

Average values of glucose in the studied groups (mg/dl)

Lun

a / m

onth

Nr.

Indi

v. /

no.

Indi

v. Valori

normale/ Normal

value

Lot martor

sănătos / Healthy control group


sicked group


treated with Siofor

Lot tratat cu

Eridiarom / Group treated

with Eridiarom


treated with Diavit

2 5 75 - 135 77,66 122,33 82 140,33 152

3 7 50 - 135 83,57 127,85 130,57 136,97

5 6 50 -135 72 183,2 124,25 121,4

7 6 50 - 135 80 203,5 129,28 126,4

Glucose is the most important sugars found in organs and normal human

blood and is the most precious fuel energy needed to achieve its work. From burning a gram of glucose by the body, resulting 4 calories.

Following the dynamics of glucose during the experimental period we see that it remained at the lower limit in healthy control group ranging between 72 and 83.57 mg / dl.

The patient group remained constant with a gradual increase from 122.33 mg / dl at 2 months climbed to 127.85 mg / dl in three months, then 5 months to 183.25 mg and 203.5 mg / dl 7 months (as the disease becomes chronic).

CREATININE

Creatinine synthesis is the liver and kidneys. Creatinine, and urea, is excreted in urine. Both substances are constant physiological concentrations in the blood which they contain. The concentration of creatinine in urine can be used as a benchmark for the excretion of analytes (albumin, alpha-amylase).



Table 3 Average values of creatinine in groups followed for 7 months (mg/dl)

Lun

a / m

onth

Nr.

Indi

v. /

no.

Indi

v. Valori

normale/ Normal

value

Lot martor sănătos

/ Healthy control group


sicked group


treated with Siofor



Lot tratat cu Diavit /

Group treated with

Diavit

2 5 0.2 - 0.8 0.553 0.593 0.450 0.463 0.497

3 7 0.2 - 0.8 0.619 0.427 0.581 0.577

5 6 0.5 – 1.1 0.515 0.193 0.555 0.518

7 6 0.2 - 0.8 0.401 0.468 0.485 0.386

After 5 months we see that the group is set sound at the lower limit of normal

creatine (0.515 mg / dl) in untreated patient group greatly decreases reaching 0.193 mg / dl and the other two lots creatinine increased slightly but remains borderline being 0.618 mg / dl in the group treated with Diavit and 0.655 mg / dl in the group treated with Eridiarom.

RESEARCH AND RESULTS INDICATE HAEMATOLOGICAL

WHITE BLOOD CELL COUNT (WBC) Table 4

Average values of leukocytes in the seven months, in the studied groups (103/µl)

Lun

a / m

onth

Nr.

Indi

v. /

no.

Indi

v. Valori

normale/ Normal

value

Lot martor

sănătos /Healthy control group

Lot bolnav

netratat / Untreated sicked group


treated with Siofor




treated with Diavit

1 6 4,1 - 10,9 7 9,75 7,58 6,52 6,82

2 5 4,1 - 10,9 7,23 9,7 8,4 7,3 6,83

3 7 2,1 – 19,5 10,21 15,34 8,25 7,32

5 6 2,1 – 19,5 10,2 13,67 7,93 6,7

7 6 2,1 – 19,5 11,91 12,76 8,5 7,89



Is slightly increased leukocyte dynamics in healthy group, significantly increased the untreated patient group remains close to normal in the group treated with Diavit.

The group treated with dynamic Eridiarom is rising up to 5 months to 7 months and then suddenly reaching down to the amount of the first month. The lowest values recorded in the group treated with Diavit.

Table 5

Average values of hemoglobin, in the studied groups (g/dl)

Lun

a / m

onth

Nr.

Indi

v. /

no.

Indi

v. Valori

normale/ Normal

value

Lot martor

sănătos / Healthy control group

Lot bolnav

netratat / Untreated sicked group

Lot bolnav tratat cu Siofor / Group treated

with Siofor



Lot tratat cu Diavit /

Group treated with

Diavit

1 6 14 - 18 14,56 13,56 14,6 15,16 14,7

2 5 14 - 18 14,43 13,6 14,7 15,26 14,56

3 7 14 - 18 15,49 16,35 15,38 18,49

5 6 14 - 18 16,06 15,83 15,55 16,14

7 6 14 - 18 16,43 15,45 16,56 19,4

During the experiment dynamics sharp increase hemoglobin from 14.7 g / dl

to 19.4 g / dl in the group treated with Diavit, very easy to grow a healthy batch of 14.56 g / dl to 16.43 g / dl and very little the sick untreated group at 13.56 g / dl to 15.45 g / dl. The group treated with Eridiarom remains almost constant increase from 15.16 g / dl to 16.56 g / dl.



ANALYSIS AND INTERPRETATION OF STATISTICAL INDICATOR BIOCHEMICAL

Table 6 The values of statistical indicators for glucose, compared to each of the groups

formed in the experiment

Loturi / Groups

Lun

a /

Mon

ths Media

(X) Average

Eroarea standard a mediei (Sx)

Average standard error

Varianţa (S2)

Variance

Deviaţia standard

(S) Standard deviation

Coeficient de variaţie

V % Coefficientof variation

Martor sănătos / Healthy control

2 14,433 0,463 0,643 0,802 5,557 5 16,063 0,159 0,101 0,318 1,980 7 16,435 0,187 0,279 0,528 3,214

Martor bolnav / Diseased control

2 13,6 0,265 0,210 0,458 3,370 5 15,835 0,093 0,034 0,185 1,168 7 15,453 0,139 0,155 0,394 2,548

Îmbolnăvit şi tratat cu Eridiarom / Diseased and treated with Eridiarom

2 15,267 0,088 0,023 0,190 1,001 5 15,555 0,318 0,405 0,636 4,089 7 16,566 0,276 0,609 0,780 4,710

Îmbolnăvit şi tratat cu Diavit / Diseased and treated with Diavit

2 14,567 0,296 0,263 0,513 3,523 5 16,148 1,050 4,412 2,100 13,008 7 19,405 0,543 2,355 1,535 7,908

Induction of diabetes with streptozotocin causes a significant increase in average blood glucose to control group, 183.25 mg respectively at 2 months and 203.5 ‰ ‰ mg to 7 months after administration. In treatment 2 months Diavit Eridiarom and a decrease in average blood glucose appears to value 57.33 mg ‰ and 52 ‰ mg. At 7 months after administration, the treatment Diavit downward trend of average blood glucose is greater, 126 405 mg ‰ to 7 months.



ANALYSIS AND INTERPRETATION OF STATISTICAL INDICATOR HAEMATOLOGICAL

HEMOGLOBIN (HGB)

Table 7 The values of statistical indicators for hemoglobin, compared to each of the groups

formed in the experiment

Loturi / Groups L

una

/ M

onth

s Media (X)

Average

Eroarea standard a mediei (Sx)

Average standard error

Varianţa (S2)

Variance

Deviaţia standard

(S) Standard deviation

Coeficient de variaţie

V % Coefficientof variation

Martor sănătos / Healthy control

2 14,433 0,463 0,643 0,802 5,557 5 16,063 0,159 0,101 0,318 1,980 7 16,435 0,187 0,279 0,528 3,214

Martor bolnav / Diseased control

2 13,6 0,265 0,210 0,458 3,370 5 15,835 0,093 0,034 0,185 1,168 7 15,453 0,139 0,155 0,394 2,548

Îmbolnăvit şi tratat cu Eridiarom / Diseased and treated with Eridiarom

2 15,267 0,088 0,023 0,190 1,001 5 15,555 0,318 0,405 0,636 4,089 7 16,566 0,276 0,609 0,780 4,710

Îmbolnăvit şi tratat cu Diavit / Diseased and treated with Diavit

2 14,567 0,296 0,263 0,513 3,523 5 16,148 1,050 4,412 2,100 13,008 7 19,405 0,543 2,355 1,535 7,908

Hemoglobin tends to increase in plots treated 15,267 g / l to 2 months at 16,566 g / l at 7 months Eridiarom treatment, 14,567 g / l to 2 months to 19.40 g / l following treatment with Diavit 5 months.

Morar et al. (1993) communicated increases in hemoglobin in treated groups compared with control group Eridiarom the experiences made on lambs and piglets

RESEARCH RESULTS BIOCHEMICAL PARAMETERS

The research were performed on 56 heads of male rats Wistar race two months old, who have subclinical diabetes caused by ip administration of 4 mg/100 body weight streptozotocin. After administration have left animals in a common lot, after 3 days I made the 4 experimental groups (separate healthy control group of 7 heads). Blood samples were collected monthly from the internal angle of the eye from 7-8 animals in each group. Blood (1.5 - 2 ml) was collected in special containers, store 4oC and immediately was sent to specialized laboratory. All tests were performed by specific enzymatic colorimetric methods to automatic biochemical analyzer - Immunology Konelab.



Total serum calcium - to consignments treated Eridiarom Diavit or dynamic to be increasing even after the first month of treatment, after two months, the increase is significant, reaching 5-7 months limit of normal data from the literature and witness healthy. The untreated patient group, after three months there is a drastic drop Like that has not recovered.

Dynamics magnesium - After 2 months of treatment in all groups, values of are small, below the lower limit of normal even in healthy group and after 3 months of treatment the values of growing up to the upper limit of normal in the group treated with Diavit and Eridiarom.

Dynamics of glucose is consistently elevated in untreated patient group and increases as the disease progresses. The plots treated with glucose dynamics Diavit Eridiarom or decreases gradually, reaching normal values after 3 months of treatment.

Serum urea - After the first¬ months of treatment in all groups illness, serum urea was elevated over a maximum even in healthy witness. After 3 months of treatment, they decreased to normal values and remained weak in plots treated with media reaching Diavit Eridiarom or literature data in the healthy control group.

Creatinine - After 2 months of¬ treatment the highest values recorded them sick untreated group and lower values in plots treated with SIOFA, Eridiarom and Diavit. After 7 months of observation, the highest values recorded them sick untreated group and lower values in plots treated with Eridiarom, Diavit and healthy controls, they reached normal limits. Overall we see that the normalization of biochemical parameters, results are

obtained within the first month of treatment (Ca), magnesium in - after 2 months of treatment, time after which values remain good and very good. These results confirm the need to monitor biochemical and hematological parameters for a long time (seven months).

Statistical processing of data confirms the biochemical results. RESULTS HAEMATOLOGICAL PARAMETERS

WBC - The untreated patient group, the value obtained is the largest and the smallest recorded in the group treated with Eridiarom and Diavit. After 3 and 5 months of treatment, but differences remain elevated in untreated patient group (9%). At 5 months, the values recorded in treated plots and Diavit Eridiarm are lower than the healthy control group and the patient group are very high.

At 7 months of observation the lowest values recorded in treated groups and the group Diavit Eridiarom and untreated patient is very high average. Lymphocytes - After 7 months of treatment a decrease of lymphocytes from untreated patient group but also to witness healthy and very significant increase in their value in the group treated with Diavit.



The healthy group values remain significantly lower then 5 months to 7 months.

Hemoglobin

After the first month of treatment, there is a slight increase in hemoglobin in the group treated with Eridiarom and treated at an important decrease Diavit the untreated control group.

After 2 months of treatment only patient group treated hemoglobin is below the average low.

After 3 months of treatment differences is emphasized in the group treated with Diavit, the average value exceeds the upper limit of the species, the same record to 5 and 7 months of observation.

At 7 months, mean hemoglobin values reach large and the group treated with Eridiarom. The lowest value recorded by the untreated patient group. So strong growth dynamics of hemoglobin in the group treated with Diavit very least healthy group and the untreated patient group. The group treated with Eridiarom remains almost constant, high values, from 2 months to 7 months.

Statistical processing of data confirms the significance of these findings. Haematocrit

After the first month of treatment hematocrit decreases the untreated patient group and the group treated with SIOFA and increase the group treated with Eridiarom. After 2 months of treatment, hematocrit values remain high and the group treated with the treated Eridiarom Diavit.

5 months to 3 respectively hematocrit value falls to the group treated with Eridiarom and maintain the lots slightly increased Diavit treated and untreated control.

After 7 months of treatment, hematocrit values are higher in the group treated with Eridiarom and Diavit, higher values than the untreated control group of healthy and sick who experience the lowest average.

Mean values are confirmed by statistical processing of data. Platelets

After the first two months of the start of the experiment, mean platelet decrease to the control group in all groups.

After 3 months, mean platelet skyrocketed untreated patient group and the group treated with media Diavit reach the average healthy control group. After 7 months of observation, mean platelet Eridiarom treated to lots and lot average Diavit healthy approach is very high but untreated patient group. Here, statistical processing of data confirms the mean value obtained along the experiment.

Following the experiment we observe that the improvement (normalization) parameter values follow the approach or untreated healthy batch of classic literature



average values after 2-3 months of observation (leukocytes, lymphocytes, hematocrit) for some parameters for their normalization must pass five or even seven months of treatment (platelets).

RESULTS HISTOPATHOLOGY Histopathological data obtained correlate with the results found in the first

months of the disease in experimental animals. Both urea and creatinine are high in the first three months of illness and then normalized.

Animal body fights to maintain normal homeostasis during the first 3-4 months of observation.

The kidney is normal healthy control group, renal glomeruli is functional is composed of a bundle of capillaries with more opportunities.

Fig. 1 Normal functioning kidney. Renal Glomerul function consists of a bundle of capillaries with more opportunities

Histopathological appearance of the kidney in diabetics and untreated group is: - The ball vascular atrophy with hypoplasia of mesangial cells and exaggerated expansion and swelling of Bowman capsule and the capsule began sclerosis; - Mesangial cells reduce their cytoplasmic area; - Reduce their long capillaries the ball vascular lumen discontinuity occurring from place to place.



Fig. 2 – Kidney patient group untreated (HE x 100) (original)

Fig. 3 Kydney, Eridiarom treated group (HE x200) (original)



Fig. 4 Rinichi lot tratat cu Eridiarom (HE x200) (original) Kydney, Eridiarom treated group

Fig. 5 Rinichi lot cu diabet netratat (după 7 luni). Glomerul atrofiat şi dilatarea

spaţiului Bowman (HE x 200) (original) Kidney untreated diabetic group (after 7 months). Glomerata atrophied and dilated

Bowman space



SELECTIVE BIBLIOGRAPHY

1. Blendea, M., Carmen P. Orbai, G. Hazi, I. Gozariu (1998) - Insulin secretion and glucose tolerance in experimental thyrotoxicosis. Rom. i. Endocrinol, 36, 3-4, 113-121.

2. Bologh Sămăghiţan V. (2004) – Modificări ionice la copii cu diabet zaharat de tip I sub tratament cu Eridiarom, Conferinţa Fundaţiei de Laborator Clinic, Oradea, 19 – 22 mai, p. 26, publicată integral în: Diabetul – alternative fitoterapeutice, 2007, p. 105 – 115.

3. Brukner I. (1980) – Medicină internă. Vol. II, Editura Medicală, Bucureşti. 4. Ciobanu Luminiţa (1988) - Eficacitatea tratamentului cu Eridiarom în

enterocolitele acute – Teză de doctorat, conducător ştiinţific Prof. Dr. N. Miu, U.M.F. Cluj-Napoca.

5. Morar R., Dana Pusta, Zoiţa Gabriela Zah, I. Paşca, O. Rotaru (2011) – Modificări histopatologice ale rinichiului de şobolan în diabetul experimental provocat cu Streptozotocină, Al XI-lea Congres Naţional de Medicină Veterinară, Bucureşti, 8 – 11 mai, p. 74.

6. Morar R., Dana Pusta, Zoiţa Gabriela Zah, I. Paşca, O. Rotaru (2011) – Cercetări asupra modificărilor histopatologice ale rinichiului de şobolan în diabetul experimental provocat cu Streptozotocină (Sz), Al XI-lea Congres Naţional de Medicină Veterinară, Bucureşti, 8 – 11 mai, poster.

Cercetări privind indicii morfo-fiziologici şi ... · Cercetări privind indicii morfo-fiziologici şi modificările rinichiului în diabetul experimental la şobolanii din linia

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