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Summary
Abstract
Introduction
Protocol
Resultados
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Medicina

Transdermal måling af glomerulær filtreringshastighed hos mekanisk ventilerede smågrise

Published: September 13, 2022
doi:
10.3791/64413
, , , , ,
1Department of Physiology,College of Medicine University of Tennessee Health Science Center

Summary

Glomerulær filtreringshastighed (GFR) er den ideelle markør til vurdering af nyrefunktionen. Standardmålemetoden ved anvendelse af inulininjektion med seriel blod- og urinanalyse er imidlertid upraktisk. Denne artikel beskriver en praktisk metode til måling af GFR transdermalt hos smågrise.

Abstract

Transdermal måling af glomerulær filtreringshastighed (GFR) er blevet brugt til at evaluere nyrefunktionen hos bevidste dyr. Denne teknik er veletableret hos gnavere til at studere akut nyreskade og kronisk nyresygdom. GFR-måling ved hjælp af det transdermale system er imidlertid ikke blevet valideret hos svin, en art med et nyresystem, der ligner mennesker. Derfor undersøgte vi effekten af sepsis på transdermal GFR hos bedøvede og mekanisk ventilerede neonatale grise. Polymikrobiel sepsis blev induceret ved cecal ligering og punktering (CLP). Det transdermale GFR-målesystem bestående af en miniaturiseret fluorescenssensor blev fastgjort til grisens barberede hud for at bestemme clearance af fluorescein-isothiocyanat (FITC) konjugeret sinistrin, et intravenøst injiceret GFR-sporstof. Vores resultater viser, at serumkreatinin steg ved 12 timer efter CLP med et fald i GFR. Denne undersøgelse viser for første gang nytten af den transdermale GFR-tilgang til bestemmelse af nyrefunktionen hos mekanisk ventilerede, neonatale grise.

Introduction

En praktisk og kvantitativ evaluering af nyrefunktionen er den glomerulære filtreringshastighed (GFR) måling, som fortæller, hvor godt nyrerne filtrerer blod baseret på clearanceprincippet1. En tidligere metode til måling af GFR indebærer intravenøs injektion af eksogene forbindelser såsom inulin eller sinistrin, der udfører serielle målinger af plasma-/urinniveauer for at detektere deres clearance 2,3. Denne metode er besværlig og kræver seriel indsamling af plasma- og urinprøver4. Et alternativ er måling af endogene metaboliske slutprodukter såsom kreatinin. Dette er imidlertid tidskrævende og til tider unøjagtigt, da det ikke kun filtreres af glomerulus, men også udskilles af tubuli 5,6. Desuden påvirkes kreatininniveauet af køn, alder, kost og muskelmasse 7,8,9.

Et mere præcist, minimalt invasivt og udbredt mål for GFR er brugen af transdermale GFR-monitorer, der måler GFR i realtid hos dyr 4,10. Sinistrin, en meget opløselig og frit filtreret eksogen nyremarkør, er mærket med fluorescein-isothiocyanat (FITC). Denne konjugerede forbindelse injiceres intravenøst, og nyrefunktionen i realtid kan vurderes uden at indsamle blod- og urinprøver11. Brugen af transdermal GFR-måling er blevet valideret hos gnavere 12, hunde13 og katte14, men ikke hos svin.

Svinearter deler flere anatomiske og fysiologiske egenskaber med mennesker, hvilket gør dem til ideelle dyr til at studere forskellige menneskelige sygdomme15. Brugen af svin i translationel biomedicinsk forskning er blevet stadig mere populær og foretrukket frem for gnavermodeller, fordi den efterligner menneskelig fysiologi og patofysiologi16. Neonatale grise er af interesse for at forstå mekanismerne for sygdomme, der er unikke for pædiatriske patienter17. Desuden sætter den nylige udvikling inden for gris til human organtransplantation en trang til at udvide de diagnostiske værktøjer til prækliniske og kliniske forsøg 18,19,20,21. Dette papir giver for første gang en vejledning til brugen af den transdermale enhed til måling af GFR hos neonatale grise.

Protocol

Procedurerne er skrevet i henhold til nationale standarder for pleje og brug af forsøgsdyr og blev godkendt af Institutional Animal Care and Use Committee (IACUC) ved University of Tennessee Health Science Center (UTHSC). BEMÆRK: Smågrise i forsøgsgruppen udsættes for cecal ligation og punktering, mens sham-gruppen kun gennemgår åbning af maven uden cecal ligation eller punktering. Smågrise i begge grupper holdes under anæstesi i 12 timer efter proceduren for at give tilstrækkelig ti…

Representative Results

I dette afsnit præsenterer vi for første gang de repræsentative data fra brugen af transdermal GFR hos neonatale grise. Vi brugte en cecal ligation og punktering model, som tidligere har vist sig at nedsætte nyrefunktionen28. Derfor antog vi, at der hos vores CLP-grise skulle være et akut fald i GFR svarende til AKI, og dette skulle detekteres på den transdermale GFR-enhed som øget clearancetid (t1/2) og derved validere dets anvendelse til svin. Syv hangrise var inkluderet, tre f…

Discussion

Dette papir beskriver praktiske trin til bestemmelse af nyrefunktion hos svin ved hjælp af de miniaturiserede transdermale GFR-skærme og FITC-sinistrin i en mekanisk ventileret, bedøvet neonatal grisemodel. Tidligere papirer har etableret eksperimentelle transdermale GFR-protokoller hos gnavere11,12,14, men der findes ingen protokoller hos svin.

For nylig har der været et drev til at udforske alte…

Declarações

The authors have nothing to disclose.

Acknowledgements

Denne undersøgelse blev støttet af National Institutes of Health tilskud R01 DK120595 og R01 DK127625 tildelt Dr. Adebiyi. Indholdet af dette papir er udelukkende forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter fra National Institutes of Health. Tak til Dr. Daniel Schock-Kusch, site director hos MediBeacon GmbH, for hans råd.

Materials

Alpha – Chloralose Sigma-Aldrich C0128-25G Used for maintanining anesthesia
Black braided silk  3-0 Surgical Specialties SP117 Silk tie for blood vessel traction and ligation
Centrifugation machine AccuSpin 8C Fischer Scientific 75-008-821 Used to extract plasma from whole blood sample
Endotracheal Tube 3.0 uncuffed Progressive Medical International 1109021995 Inserted through tracheostomy
FITC-Sinistrin 1.0 g MediBeacon Inc. FTCF S001 Store at room temp and protect from light
GEM Premier 3000 Blood gas analyzer Instrumentation Laboratory 5700 For bedside blood gas analysis
Heating Pad medium size 20 in x 29 in Adroit Medical Systems V029 Connects to heat therapy pump
HTP-Heat Therapy Pump Adroit Medical Systems HTP Allows you to set temperature as needed.
IDEXX Catalyst One IDEXX Laboratories 89-92525-00 Plasma creatinine analysis
Invasive blood pressure catheter 3.5Fr Millar SPR-524 Inserted in femoral artery
IV adminstration set with flow regulator True Care TCRTCBINF033G Used to connect IV fluid bag to vein catheter
Ketamine Covetrus 68317 Used for induction of Anesthesia
MediBeacon analysis software version 3.0 MediBeacon Inc. N/A Software program used for analysing data to obtain sinistrin clearance half life and curve
Millex-GV Syringe Filter Unit 0.22 µm Millipore Sigma SLGVR33RS Syringe filter for chloralose injection
Neonate/Infant Ventilator Sechrist Millennium 20409 Connected to air supply to provide ventilation through endotracheal tube
Phenobarbital Sodium + Phenytoin Sodium (Euthasol) Covetrus 72934 Used for euthanasia
Ringer Lactate 500 mL bag Baxter 2B2323Q Maintanence fluid infusion
Sterile Gloves Henry Schein 104-5920 Used by operator during surgery
Sterile Gown Halyard Health 95021 Used by operator during surgery
Steril Towel Medline 42131704 Used as drape to maintaine sterile field when operating
Suture 3-0 silk reverse cutting needle Ethicon NC1842168 Used for suturing abdominal wall layers
Transdermal Mini GFR Monitor MediBeacon Inc. TDM004 Battery and USB connector included in package
Transdermal monitor adhesive patch MediBeacon Inc. PTC-SM001 Doubl sided adhesive patch for GFR probe
Umbilical Tape 1/8 in x 20 yds Fisher Scientific NC9303017 To secure endotracheal tube
Venous Catheter size PE/5 Micro medical tubing BB31695 For femoral vein cannulation
Xylazine Covetrus 61035 Used for induction of anesthesia
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Tags

Keywords: Transdermal GFR MeasurementKidney FunctionSwine ModelXenotransplantationPigletsCecal LigationTracheostomyVessel CannulationReal-time AssessmentNon-invasive
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Fanous, M. S., Afolabi, J. M., Michael, O. S., Falayi, O. O., Iwhiwhu, S. A., Adebiyi, A. Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets. J. Vis. Exp. (187), e64413, doi:10.3791/64413 (2022).
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