HOT LAB

HANDS ON THE LAB

Model Hewan Coba

Nur Arfian
Departemen Anatomi FKUGM
 MODEL HEWAN COBA
• Mimic pathological condition in human(but
different)
• Cheap….use suture / clamp
• High predictive results (pathological): fibrosis,
inflammation, tubular injury, neo-intimal
formation
• Evaluate the result: simple isbetter
• Genetic modification : is it needed?
– Atherosclerosis
 Model hewan coba
• Frequently selected because of their similarity to
humans in terms ofgenetics, anatomy, and physiology.
• Unlimited supply and ease of manipulation.
– Kontrol variabel mudah
– Bias tidak besar (background yg sama, BBsama, perlakuan
sama)
• Therefore, scientists cannot conduct research on just
one animal or human, and it is easier for scientists to
use sufficiently large numbers of animals (rather than
people) to attain significantresults.
 Ligating and clamping
• Ligating: Model for obstruction, lead to
fibrosis in ureteral ligation or bile duct ligation
or neointimal formation/early injury of
atherosclerosis in carotid/femoral arteries.
• Destructive of parenchymal,inflammation
(sterile) and Fibrosis
– Dilihat secara genomik-proteomik
– Seluler : Histopatologi
Unilateral Ureteral Obstruction; Kidney fibrosis
 Clamping
• Pada pembuluh darah.
• Model ischemic-reperfusi: periode iskemia
(hentinya aliran darah selama bbrp menit, 20-
30 an menit pada mencit); dilanjutkan periode
reperfusi (kembalinya pembuluh darah)
• Stenosis…clip (arteri renalis, aorta: dengan
tali)
• Rusaknya parenkhim karena iskemia
 Model kidney Ischemic/reperfusion injury

Arfian, et al, Under revision in

Biomed Research International (Q1)
Bilateral carotid arteries clamping

C2: BCACfor 20 minutes

EJN, Q1accepted
 How to choose….
• Depend on our purpose of study
• Specific pathomecanism
• Spectrum of the model:
– Carotid ligation: neointimal formation butnot
atherosclerosis
– Kidney fibrosis: not for functionalmeasurement
(creatinin)
– Subtotal nephrectomy: chronic kidney diseases
• Method of research thatwe master
• Method for evaluation
 Penyakit Ginjal
MODEL
Glomerulu
GLOMERULOSCLEROSIS
Tubulo-interstisial s 80% -Diabetic Nephropathy
20% -filtrasi -FSGS(Adriamycin)

MODEL MODEL
GAGALGINJALAKUT GAGALGINJALKRONIS
-Kidney Ischemic reperfusion injury (IR) -5/6 SubtotalNephrectomy
-Nephrotoxic (cyclosporin dll) -Diabetic nephropathy
-Urate/Hyperuriemia nephropathy
-Hipertension related Nephropathy
-High protein diet
KIDNEY FIBROSIS
- Unilateral Ureteral Obstruction
 Experimental models in renal injury
Filtration disruption
• Glomerulosclerosis
– Adriamycin nephropathy (FSGS)
• Diabetic nephropathy
Tubulointerstisial injury
• ischemic-reperfusion injury model : acute kidney injury model
– Bilateral Renal pedicles clamping
• Renal artery stenosis
– Hypertension induced renal failure
• Nephrotoxic agent: cyclosporin, COXinhibitor
• Hyperprotein diet
Fibrosis : Unilateral ureteral ligation
Hypertension: Renal artery stenosis , High salt inducedhypertension/renal
injury (uninephrectomyzed combined)
• Gabungan
– Renal ablation (5/6 sub-total nephrectomy)
Model gagal Ginjal Akut
The 5/6 subtotal nephrectomy procedure was performed after anaesthesia with a 0,1
mL/10gBW i.p. pentobarbital injection. In this model, we performed a uninephrectomy on
the right kidney and 2/3 of the remaining kidney was ablated by a polar excision at 1 day
after the uninephrectomy.
(Ni Putu Nita, et all. Under revision, published anywhere)
 Model Hewan Coba peny. Ginjal
dengan operasi
• Relatif murah….minor set, suture, anastesi
(inhalasi, Na penthobarbital, ketamin).
• Hasil bisa diprediksi dengan waktu yang
sesuai.
• Cepat dan abrupt.
• Sesuai dengan keadaan penyakit manusia
(walaupun ada perbedaannya).
 Renal Blood Flow (RBF) measurement
10

15

20

25

30

35
0

5
isch0
isch4
isch8

in Ischemic-Reperfusion Injury
isch12
isch16
isch20
isch24
isch28
isch32
isch36
isch40
isch44
isch48
isch52
isch56
isch60/10'
isch64
isch68
isch72

RBF(AU)
isch76
isch80
isch84
reperfs 1
reperfs 5
reperfs 9
reperfs 13
reperfs 17
reperfs 21
reperfs 25
reperfs 29
reperfs 33
reperfs 37
RBF(AU)

reperfs 41
reperfs 45
reperfs 49
reperfs 53
reperfs 57
reperfs 61
reperfs 65
reperfs 69
reperfs 73
reperfs 77
reperfs 81
reperfs 85
 Kidney Ischemia/Reperfusion Injury (IRI)
MODEL: PUNYASCOPE Delayed epithelial injury,
Kidney fibrosis,
40% followed by chronic renal failure
Epithelial cells, polarity loss
as the main actor Ischemic/ apoptosis
reperfusion dedifferentiation
TLR-4 in-adequate CGA
TLR-4 Recovery
MCP-1 TLR-4 TLR-4
TLR-4 MCP-1
MCP-1 MCP-1
MCP-1

PERICYTE CGA
TGF-β1
fIBROBLAST TGF-β1
TGF-β1
TGF-β1

myofibrobast Macrophage infiltration

Matrix deposition Peritubular capillary loss

Kidney Ischemic reperfusion
 kerusakan glycocalyx / lapisan permukaan endothelial setelah
cedera iskemik/reperfusion pada arteri intra-renal.

Sel
endothelial / Glycocalyx /
vW Factor WGA OVERLAY αSMAgreen
SHAM
D1 /R

(Arfian, KnE Life Science 2015)

 CKD dan Kidney Fibrosis

Initial Causes :
Hypertension
Diabetes
Chronic Kidney Diseases
Immune Diseases (CKDs)
Glomerulonephritis
Ischemic

Kidney Fibrosis
- Kidney fibrosis is regarded as the final common pathway for most forms
of progressive renal disease,
including glomerulosclerosis & tubulo-interstitial fibrosis.
Ginjal normal vs ginjal CKD / Fibrosis (Ligasi
pada ureter / Unilateral Ureteral Obstruction)
Fibrotic Kidney
Normal Kidney

myofibroblast Macrophage infiltration Robert L. Chevalier; 2009

Won Kim, et all; 2006
Matrix deposition Peritubular capillary loss
 Models progressivitas –(UUO)
• leading within 24 h to reduced renal blood flow and
glomerular filtration rate
• followed within several days by hydronephrosis,
interstitial inflammatory infiltration (macrophages),
and tubular cell death attributable to apoptosis and
necrosis.
• progression to a severely hydronephrotic kidney
with marked loss of renal parenchyma takes place
over 1–2 weeks, with more severe fibrosis in the
neonate than the adult
Perubahan Histopatologis Skematik
Makroanatomi UUO
Time after time…..
Day7 of UUO

Day14 of UUO Day14 of UUO

Histopatologis Ginjal UUO
Nephrectomy
• Cutting the poles
– Bleeding
– R. superior dan inferior A.
Renalis ligation???
– Electrocauter meningkatkan
keberhasilan
– Antiseptic
– Bisa dikolaborasikan dgn
diet tinggi garam: model
hipertensi
Simvastatin in reducing CKD(5/6 Subtotal nephrectomy) model

Ni Putu Nita, Ngatidjan, Arfian, 2015.IJPPS2016

 Examine the result…
• Functional:
– Blood: Creatinine, biomarker
– Urine: proteinuria, glukosa,albumin
• Histopathology: Basic staining
– Glomerulosclerosis: scoring, PASor HE
– Tubular Injury: scoring, PASor HE
– Fibrosis: fraction area, Sirius Red or massonTrichome
– Vessel (remodeling, hyalinosis): wall thickness
• Immunohistochemical staining (IHC)
– Fraksi area (fibrosis, myofibroblast)
– Jumlah Sel
– Ekspresi
• DNA-RNA-Protein
– realtimePCR; PCR
– Westernblotting, ELISA
Pengukuran variable Histopatologi
• Skoring
– Penetapan karakteristik penilaian
– Penetapan skor
• Pengukuran Luas
– Software ImageJ
• Pengukuran fraksi
– Dalam bentuk persen
– Berapa persen area positif yg terwarnai dalam satu lapang
pandnag
• Pengukuran volume
– Stereologi
Pengukuran Genomik-Proteomik
 Tubulo-intertitial injury
• Twenty fields of tubulointerstitial area inthe
• cortex were observed and graded asfollowing:
• 0, normal;
• 1, the area of interstitial inflammation and fibrosis,
tubular atrophy, anddilation
• with cast formation involving ,25% of thefield;
• 2, lesion area between 25% and 50% of the field; and
• 3, lesions involving 50%-75% of the field.
• 4, lesions involving >75% of the field
• (Hypertension 2000;36;561-568)
Ginjal normal
 Cedera tubulus
-temukan perbedaannya dgn yg normal?-
Day 14 of UUO
I/R Reperfusion Normal
 Glomerulosclerosis index, from PAS
staining
• Based on PASstaining
• Histopahology examination of kidney
(glomerulus)
– Adhesion of glomerular tuft and bowman capsule
– Mesangial expansion and matrix accumulation
– Glomerular tuft obstruction
– cellular expansion in bowman space
 Dasar penilaian
• 0, normal;
• 1,slight glomerular damage, the mesangial
matrix and/or hyalinosis with focaladhesion,
involving ,25% of the glomerulus;
• 2, sclerosis of 25% to 50%;
• 3, sclerosis of 50% to 75%;and
• 4, sclerosis of .75% of the glomerulus
 Tubulo-intertitial injury
• Twenty fields of tubulointerstitial area inthe
• cortex were observed and graded asfollowing:
• 0, normal;
• 1, the area of interstitial inflammation and fibrosis,
tubular atrophy, anddilation
• with cast formation involving ,25% of thefield;
• 2, lesion area between 25% and 50% of the field; and
• 3, lesions involving 50%-75% of the field.
• 4, lesions involving >75% of the field
• (Hypertension 2000;36;561-568)
Ginjal normal
 Cedera tubulus
-temukan perbedaannya dgn yg normal?-
DIT: Diffuse
intimal Thickening

Lesion type 1
Lipid extracellular,
Mild macrophage

Lesion type 2
Foam cells,
Non-foamy macrophage
 Model of Atherosclerosis
• Different step of atherosclerosisbetween
human & animal.
• Model of subintimal thickeningatau
neointimal formation
– Carotid ligation
– Represent early injury of atherosclerosis
– Inflammation with Macrophageinfiltration
– Subintimal thickening of artery
Atherosclerosis in Human dan rodents
Histopatologi
 Variabel pada penelitian
• Apa saja ynag dihitung???
• Histopathologi
– Morfologi
– Scoring: penetapan suatu kriteria, skor dalam lapangpandang
– Fraksi area: fibrosis
– Luas, tebal, ukuran: luka, luas lumen, tebal epithel
• Ekspresi
– Kembali ke dogma sentral DNA RNAProtein
– IHC? Bisakah utk mengukur ekspresi?
• Fungsional
– Darah
– Uji khusus : memori, ROS
• Uji khusus
 Setting penelitian
• Memeriksa efek Clorogenic acid (CGA) thd cedera iskemik/reperfusi
ginjal pada mencit
• Model Gagal Ginjal Akut/AKI
• Clamping pediculus renalis (A et V Renalis) selama 30 menit,
reperfusi
• Mencit (3-4 bulan) dibuat model cedera I/R ginjal, kemudian
diberikan CGAselama 2 hari, kemudianditerminasi.
• Memeriksa efek CGAthd:
– Cedera tubulus
– Proliferasi sel epithelial
– Proses inflamasi
– Fibrosis

– Apa yang akan kalian periksa?

 Kidney Ischemia/Reperfusion Injury (IRI)
Delayed epithelial injury,
Kidney fibrosis,
40% followed by chronic renal failure
Epithelial cells, polarity loss
as the main actor Ischemic/ apoptosis
reperfusion dedifferentiation
TLR-4 in-adequate
TLR-4 Recovery
MCP-1 TLR-4 TLR-4
TLR-4 MCP-1
MCP-1 MCP-1
MCP-1

PERICYTE
TGF-β1
fIBROBLAST TGF-β1
TGF-β1
TGF-β1

myofibrobast Macrophage infiltration

Matrix deposition Peritubular capillary loss

 Banyak ukuran yg kita lakukan
• Histo pathologi
• Ekspresi
– DNA……..RNA…….Protein
• Fungsional
– Fungsi ginjal, hepar, jantung
• Kimiawi
• Makro-anatomi
– Keadaan makro suatu organ (Plak, iskemia,
senescence)
Pengukuran variable Histopatologi

• Skoring
– Penetapan karakteristik penilaian
– Penetapan skor
• Pengukuran Luas
– Software ImageJ
• Pengukuran fraksi
– Dalam bentuk persen
– Berapa persen area positif yg terwarnai dalam
satu lapang pandnag
• Pengukuran volume
– Stereologi
Normal
Abnormal PAS-tubular injury SiriusRed-Fibrosis PAS- Glomerulosklerosis

Fibrosis (%) Glomerulosclerosis

15 Score
*** 4
10 ***
** 3
##
2 *** #
5 #
1
0
0
IHC - myofibroblast VESSEL IHCFibroblast IHCPodosit Kapiler IHC
Nefrin
(PDGFRβ) CD31

PERIVASCFIBROSISSCORE
Fibroblast cell number Capillary
4
Interstitial 100
(PDGFRβ positive rarefraction (%)
3
cells)
30 Fibrosis(%) 2
80
1
0 60
20 NORMAL UUO *** *** ***
40
20
Mean Wall Thickness(µm)20
10
15
0
0 10

0
Simvastatin in reducing CKDexperimental model

Ni Putu Nita, Ngatidjan, Arfian, 2015.IJPPS2016

 Hyperuricemia induced glomerulosclerosis
with podocyte injury
BmcNephro,2017
Creatinin (mg/dL)
Asam Urat (mg/dL)
1.2
5
*** 1
* PODOCIN
4
***
*
0.8 NEPHRIN
3 # #
0.6
2 GAPDH
0.4 3
1 # #
0.2 #
0 0 2.5 #

* *
2 *
1.5

A.U
.
1

0.5

Podocin/GAPDH Nefrin/GAPDH
Control AU7 AU14 AAL7 AAL14
Glomerulosclerosis
 Fibrosis dan myofibroblast
control D3 UUO
D14 UUO
Sirius Red
Alpha SMA
 Sirius Red Staining
CONTROL D14 UUO
A B E
Fibrosis Area (%)*
10
α-SMA
WT

9
8
7
TGF β
6 psmad3

(%
5
VEETKO

)
4 β actin
3
control control UUO UUO
2
1
0
Control D14 of UUO

αSMA-Myofibroblast
C D
CONTROL D14 UUO αSMA fraction area (%)
10 *
9
WT

8
7
6
(%)
5
4
3
2
VEETKO

1
0
Control D14 of UUO

Wild Type mice

VEETKO:Vascular Endothelial Endothelin-1 Knock-Out Mice with ET-1 deletion from EC
Pericyte/fibroblast αSMA (Smooth muscle /
Myofibroblast)

nuclei
…..Teams up…..
RESEARCH