Microwave processing for sample preparation to evaluate mitochondrial ultrastructural damage in hemorrhagic shock
Gary D Josephsen1, Kelly A Josephsen, Greg J Beilman
1Section of Surgical Critical Care, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA. jose0118@umn.edu
Abstract:
This is a report of the adaptation of microwave processing in the preparation of liver biopsies for transmission electron microscopy (TEM) to examine ultrastructural damage of mitochondria in the setting of metabolic stress. Hemorrhagic shock was induced in pigs via 35% total blood volume bleed and a 90-min period of shock followed by resuscitation. Hepatic biopsies were collected before shock and after resuscitation. Following collection, biopsies were processed for TEM by a rapid method involving microwave irradiation (Giberson, 2001). Samples pre- and postshock of each of two animals were viewed and scored using the mitochondrial ultrastructure scoring system (Crouser et al., 2002), a system used to quantify the severity of ultrastructural damage during shock. Results showed evidence of increased ultrastructural damage in the postshock samples, which scored 4.00 and 3.42, versus their preshock controls, which scored 1.18 and 1.27. The results of this analysis were similar to those obtained in another model of shock (Crouser et al., 2002). However, the amount of time used to process the samples was significantly shortened with methods involving microwave irradiation.
Insights
Microwave processing rapidly prepares liver biopsies for transmission electron microscopy (TEM) to assess mitochondrial damage from metabolic stress. This method significantly reduces processing time while revealing ultrastructural changes indicative of shock.
Area of Science:
- Cell Biology
- Pathology
- Biomedical Engineering
Background:
- Metabolic stress, such as hemorrhagic shock, causes significant ultrastructural damage to mitochondria.
- Accurate assessment of mitochondrial damage is crucial for understanding shock pathophysiology.
- Traditional methods for preparing samples for transmission electron microscopy (TEM) can be time-consuming.
Purpose of the Study:
- To adapt and report on the use of microwave processing for preparing liver biopsies for TEM.
- To examine the ultrastructural damage of mitochondria in liver tissue subjected to hemorrhagic shock.
- To evaluate the efficiency of microwave irradiation in accelerating sample preparation for TEM analysis.
Main Methods:
- Hemorrhagic shock was induced in pigs, with hepatic biopsies collected before and after resuscitation.
- Microwave irradiation was employed for rapid processing of liver biopsy samples for TEM.
- The mitochondrial ultrastructure scoring system was used to quantify damage in pre- and post-shock samples.
Main Results:
- Post-shock liver biopsy samples exhibited significantly increased mitochondrial ultrastructural damage compared to pre-shock samples.
- Scoring indicated a marked increase in damage severity following the shock and resuscitation period.
- Microwave irradiation significantly reduced the overall time required for sample preparation for TEM.
Conclusions:
- Microwave processing is an effective and rapid method for preparing liver biopsies for TEM analysis of mitochondrial damage.
- The study confirms the utility of TEM and a standardized scoring system in quantifying shock-induced mitochondrial injury.
- This adapted methodology offers a time-efficient approach for investigating cellular damage in metabolic stress models.
