Anaesthesia, Pain, Intensive Care and Emergency - Part 5 pptx

But all too often shock is so devastating,because the dose of stress exceeds the cell’s capacity for maintaining integrity, thatthe cellular tools are driven to induce the death of the c

ciency of the treatments appears to be tied to removal of inflammatory mediators,even though no difference in mortality between specific treatments has beenconfirmed in the literature.

More specific approaches have been proposed, such as high-volume tration and continuous plasma filtration [16, 17], in order to remove several pro-and anti-inflammatory mediators and to overcome the limitations of conventionalcontinuous renal replacement therapy (CRRT) (i.e., low volume exchange and lowsieving coefficients for sepsis-associated mediators)

haemofil-In order to improve the efficacy of a blood purification system in the criticallyill septic patients, unselective adsorption onto a cartridge was added to plasmafiltration and conventional diffusion/convection in a newly designed extracorpo-real device called coupled plasma filtration–adsorption (CPFA) (Fig 1)

CPFA is a specific method for the treatment of sepsis The equipment it requires

Fig 1 The peak concentration hypothesis suggests that nonselective control of the peaks ofinflammation and immunoparalysis may help to restore immunohomeostasis

The life of the cartridge, as demonstrated by in vitro experiments, is 10 h, whichcorresponds to the mean expected treatment duration.

In recent years resins and charcoals have been used because of their capacityand ability to remove toxic substances from blood, but the medical applicationswere often counterbalanced by safety concerns, such as leaching of metals, release

of small microparticles and poor homogeneity and biocompatibility fusion through ion/cation exchange resins was first proposed in 1948 for thetreatment of renal failure, but several variations followed Early experience andtreatments were complicated by pyrogenic reactions, electrolyte disturbances andhaemolysis

Haemoper-In fact, the use of more sophisticated technologies to coat resins reduces theproblems that result from loss of efficiency, poor reproducibility and mixed out-comes Extracorporeal applications require that resin is defined in terms of thechemical nature of the resin, particle size, porosity and surface area Resins must

be also tested for the release of microparticles, heavy metals and other toxicsubstances The resin test is done in real conditions similar to those obtainingduring a patient application The optimisation of flow and column geometry is aparameter that also greatly influences adsorption efficacy There is a balancebetween the volume of plasma being treated and the time plasma is in contact withthe resin [18]

Using an experimental model of acute endotoxaemia in rabbits, Tetta et al

Fig 2 Scheme of coupled plasma filtration–adsorption

Infusion

Haemofilter

Cartridge

Plasma Plasmafilter

studied whether nonselective adsorption from plasma of cytokines and otherpro-inflammatory mediators known to be produced in excess during sepsis couldreduce 72-h mortality Cumulative survival was significantly improved in rabbitstreated with CPFA, and cumulative survival of the resin with the lipopolysaccha-ride (LPS) group was not significantly different from that of the control group(Fig 3) [19].

Human studies are limited, but promising: Ronco et al compared CPFA againsthaemodiafiltration by measuring homodynamic and immune responsiveness inARF patients in septic shock These authors observed that the haemodynamic wassignificantly better with the use of CPFA than with haemodiafiltration They alsoobserved significantly higher leucocyte responsiveness after CPFA treatment [20].Another clinical study was conducted by Formica et al The authors examinedthe effect of repeated applications of CPFA on haemodynamic response in septicpatients with and without renal failure In this long-term study, the authors showedCPFA to be a safe and feasible treatment leading to significant improvements inhaemodynamic stability, vasopressor requirement, pulmonary function, and 28-and 90-day survival (Fig 4) The 28-day survival rate was 90%, which was quiteunexpected considering an APACHE II-predicted mortality of about 40% for thesepatients [21]

On the grounds of these experiences it was also expected that early therapywould hamper the inflammatory cascade

In the light of these remarks, GiViTI decided to launch a collaborative mised controlled trial for formal evaluation of the efficacy and clinical safety ofCPFA in septic shock The main study objective is to clarify whether the implemen-tation of CPFA in addition to the current clinical practice can reduce mortality ofseptic shock patients in ICU The second objective of the study is to determine

rando-Fig 3 Coupled plasma filtration–adsorption in a rabbit model of endotoxic shock

whether CPFA can reduce the incidence of organ dysfunction and length of stay.The study will involve Italian, adult, general ICUs affiliated to the GiViTI group,

in which CPFA is regularly used in the treatment of septic shock The study isrestricted to ICUs that, based on the promising but still incomplete evidenceavailable, have already introduced CPFA into their routine practice In other words,

we ask the staff at these centres to use CPFA within a research programme that willyield information on the real efficacy of the treatment

All patients who are admitted to the ICU in septic shock or who develop septicshock while in the ICU will be eligible The definition used for septic shock is thatprovided by the international literature [22, 23] Patients will be considered eligiblefor the study only if it will be possible to initiate CPFA in less than 6 h either fromadmission to the ICU for patients admitted in septic shock, or from the diagnosis

of septic shock for the others

There are some exclusion criteria that make patients not eligible for the study;these concern age, pregnancy, cerebral coma, metastatic cancer, cardiopulmonaryresuscitation, life expectancy, etc Eligible patients will be identified upon admis-sion or during the stay in the ICU and randomised Patients randomised to thecontrol arm will be treated according to the current clinical practice in the ICU.Patients randomised to the experimental arm will also be treated according to theICU’s current clinical practice, but with the addition of CPFA

The CPFA treatment will be applied intermittently (10 consecutive hours lowed by a 14-h break or CVVH for patients with renal failure) for 5 days followingrandomisation The cartridge must be changed after 10 h; previous experience hasshown saturation of the resin after this

fol-The clinical follow-up starts on the day of randomisation and finishes at

Fig 4 Trend in mean arterial pressure (MAP) throughout the first ten sessions (each point

is the mean of the measure at that time for all patients) Statistical significance is related tothe difference between all 100 pre- vs posttreatment measurements

discharge from the ICU During the ICU stay, information on compliance with the

four A-level recommendations of the Surviving Sepsis Campaign [24], and the daily

SOFA score (Sequential Organ Failure Assessment) [25] will be recorded The vitalstatus will be recorded at ICU discharge, at hospital discharge and at 90 days fromrandomisation For patients transferred to other hospitals, “vital status at hospitaldischarge” will be intended as the vital status at discharge from the latest hospital

in which the patients stayed

In agreement with the study rationale, lower mortality is expected in patientstreated with CPFA than in patients treated according to standard practice only Inthe light of these considerations, the following primary and secondary end-pointswere chosen:

· Mortality at hospital discharge For patients transferred to other hospitals, itwill be intended as mortality at the discharge from the latest hospital in whichthe patients stayed

· Mortality within 90 days of randomisation With this end-point it will bepossible to evaluate whether a possible benefit obtained in the short term(hospital discharge) is maintained afterwards

· Proportion of patients who develop one, two, three and four new organ failuresduring their ICU stay A new organ failure is defined as a change in SOFA scorefrom 0, 1 or 2 to 3 or 4 in any of the systems considered [26] This end-pointwill determine whether CPFA can reduce the risk that organ failures willdevelop

· Days not spent in the ICU during the first 30 days after randomisation Withthis end-point it will be possible to determine whether CPFA can reduce thecomplexity of these patients’ care

Data previously published by GiViTI show a hospital mortality rate of 63% inseptic shock patients The study is designed to reveal a 25% relative improvement

in hospital mortality with the use of CPFA For it to have a power of 80% to findout such a difference with 5% type I error, it is necessary to enrol 155 subjects ineach arm Increasing this estimate by approximately 5% to prevent possible pro-blems in compliance with the protocol yields a number of patients needed of 330.This sample allows detection of a 29% difference with a power of 90%

The trial will be monitored with the Bayesian approach As known, theBayesian approach combines a prior distribution and the gathering of theexperimental evidence into a posterior distribution The posterior distributionwill be the basis on which to decide whether to interrupt the trial or not Hence,this analysis requires a probabilistic formalisation of two conflicting hypothe-ses: one sceptical and one enthusiastic The trial will be interrupted earlier thanplanned when the patient’s benefit is achieved (i.e., demonstration of treatmentefficacy), when sceptics are convinced of the treatment efficacy or, in other words,when the posterior distribution deriving from a prior sceptical hypothesis ac-knowledges the achieved benefit Conversely, the trial will be interrupted earlierthan planned in case of treatment’s futility (i.e., demonstration that the treatment

is futile) when a prior enthusiastic approach is curbed by the treatment ness or, in other words, when the posterior distribution deriving from a prior

useless-enthusiastic hypothesis acknowledges the unchanged conditions.

Before enrolment, all patients will be given information on the study’s tives, procedures and correlated risks

objec-If any patient is not able to give consent, the instructions provided by theInternational Commission on Harmonisation will be followed (ICH Guideline forGood Clinical Practice) We consider that this trial is extremely important, to provethe effectiveness of this technique in decreasing morbidity and mortality in septicshock If we obtain a positive result we can conclude that sepsis can be treated byblood purification technology, but even if we do not, the study will still be importantbecause its result will modify the current clinical practice in ICUs

The trial has been registered with both the ClinicalTrials.gov (identifierNCT00332371) and the ISRCTN (24534559) registries

References

1 Friedman G, Silva E, Vincent JL (1998) Has mortality of septic shock changed with time?Crit Care Med 26:2078–2086

2 Wheeler AP, Bernard GR (1999) Treating patients with sepsis N Engl J Med 340:207–214

3 USA National Vital Statistics Report (2001) 49:6

4 Rossi C, BertoliniG (2005)Progetto Margherita (thesis).(Rapporto2004)Sestante, Bergamo

5 Alberti C, Brun-Buisson C, Burchardi H et al (2002) Epidemiology of sepsis andinfection in ICU patients from an international multicentre cohort study Intensive CareMed 28(2):108–121

6 Liano G, Pascual J (1996) Acute renal failure Madrid Acute Renal Failure Study Group.Lancet 17:347–349

7 Rangel-Frausto MS, Pittet D, Costigan M (1995) The natural history of the systemicinflammatory response syndrome (SIRS) A prospective study JAMA 273:117–123

8 Bellomo R, Ronco C (1998) Indications and criteria for initiating renal replacementtherapy in the intensive care unit Kidney Int 53[Suppl 66]:S106–S109

9 Kellum JA, Johnson JP, Kramer D et al (1998) Diffusive vs convective therapy: effects

on mediators of inflammation in patient with severe systemic inflammatory responsesyndrome Crit Care Med 26:1995–2000

10 Hotchkiss RS, Karl IE (2003) The pathophysiology and treatment of sepsis N Engl JMed 348(2):138–150

11 Annane D, Bellissant E, Cavaillon J-M (2005) Septic shock Lancet 365:63–78

12 Singh S, Evans TW (2006) Organ dysfunction during sepsis Intensive Care Med32(3):349–360

13 Mira JP, Cariou A, Grall F et al (1999) Association of TNF2, a TNF-alpha promoterpolymorphism, with septic shock susceptibility and mortality: a multicenter study.JAMA 282:561–568

14 Godin PJ, Buchman TG (1996) Uncoupling of biological oscillators: a complementaryhypothesis concerning the pathogenesis of multiple organ dysfunction syndrome CritCare Med 24:1107–1116

15 Wheeler AP, Bernard GR (1999) Treating patients with severe sepsis N Engl J Med340:207–214

16 Ronco C, Brendolan A, Bellomo et al (2004) The Rationale for Extracorporeal Therapies

in Sepsis Adv Sepsis 4(1):2–10

17 Bellomo R, Baldwin I, Cole L et al (1988) Preliminary experience with high volumehemofiltration in human septic shock Kidney Int 53:182–185

18 Brendolan A, Ronco C, Ricci Z et al (2004) Coupled plasma filtration adsorption:rationale, technical development and early clinical experience sepsis, kidney andmultiple organ dysfunction Contrib Nephrol 144:376–386

19 Tetta C, Gianotti L, Cavaillon JM et al (2000) Continuous plasmafiltration coupled withsorbent adsorption in a rabbit model of endotoxic shock Crit Care Med 28:1526–1533

20 Ronco C, Brendolan A, Lonnemann G et al (2002) A pilot study on coupled plasmafiltration with adsorption in septic shock Crit Care Med 30:1250–1255

21 Formica M, Olivieri C, Livigni S et al (2003) Hemodynamic response to coupledplasmafiltration–adsorption in human septic shock Intensive Care Med 29(5):703–708

22 Levy MM, Fink MP, Marshall JC et al (2003) 2001 SCCM/ESICM/ACCP/ATS/SIS national Sepsis Definition Conference Intensive Care Med 29(4):530–538

Inter-23 Bone RC, Balk RA, Cerra FB et al (1992) Definitions for sepsis and organ failure andguidelines for the use of innovative therapies in sepsis The ACCP/SCCM ConsensusConference Committee American College of Chest Physicians/Society of Critical CareMedicine Chest 101(6):1644–1655

24 Dellinger RP, Carlet JM, Masur H et al (2004) Surviving Sepsis Campaign guidelinesfor management of severe sepsis and septic shock Intensive Care Med 30(4):536–555

25 Vincent JL, de Mendonca A, Cantraine F et al (1998) Use of the SOFA score to assessthe incidence of organ dysfunction/failure in intensive care units: results of a multicen-ter, prospective study Working Group on “Sepsis-related Problems” of the EuropeanSociety of Intensive Care Medicine Crit Care Med 26(11):1793–1800

26 Iapichino G, Radrizzani D, Bertolini G et al (2001), Daily classification of the level ofcare A method to describe clinical course of illness, use of resources and quality ofintensive care assistance Intensive Care Med 27(1):131–136

CPR AND TRAUMA

The cell in shock

M.M MORALES, H PETRS-SILVA

‘Cellular homeostasis’ is any of the processes involved in the maintenance of aninternal equilibrium within a cell or between a cell and its external environment.The physical and biochemical parameters of physiological equilibrium conducive

to eukaryotic cell function include availability and maintenance of nutrients,oxygenation, temperature, pH, and osmolality, but exposure to conditions whenthese parameters are outside the physiological ranges is considered to cause stress

to the cell, leading to macromolecular damage Many types of environmental stresshave been shown to cause deleterious changes in cells, including osmotic stress [1],thermal stress [2], heavy metal stress [3], ionising radiation [4], baric stress [5],oxidative stress [6], chemical genotoxin stress [7], mechanical injury stress [8] andhypoxia/ischaemia [9]

As a reaction to the threat of macromolecular damage from sudden mental change or frequent fluctuations in environmental factors, the cell induces

environ-a stress response This response henviron-as been described environ-as environ-an evolutionenviron-arily highlyconserved mechanism of cellular protection [10] The endpoints of stress eventsinclude quick responses, such as protein modifications (e.g protein phosphoryla-tion) [11], changes in Ca2+concentrations [12], and slow responses, such as proteinchaperoning and repair, transcriptional regulation, removal of damage proteins,DNA and chromatin stabilisation and repair, cell-cycle control, cell proliferationand apoptosis [13]

Cells respond to multiple opposing signals simultaneously, and the decision onwhether to die or survive will depend on the intensity of the stress signal Anextreme condition of stress represents a cell in shock The cells have a few tools forreversing shock before it goes too far But all too often shock is so devastating,because the dose of stress exceeds the cell’s capacity for maintaining integrity, thatthe cellular tools are driven to induce the death of the cell [14–16] This process isphysiological, since it serves to avoid the genesis of tumours and genetic instability

of organisms [17]

Cellular stressors

Heat shock and the heart shock proteins

Ashburner and Bonner wrote the first review on the induction of gene activity byheat shock 27 years ago, describing how immediately after an increase in tempera-ture all cells increase production of a certain class of molecules called heat shockproteins [18] Subsequent studies have revealed that the same response takes placewhen cells are subjected to a wide variety of environmental insults, such as toxicmetals [19], alcohols [20], and many metabolic insults [21]

Similar changes in gene expression provide a rapid and direct mechanism ofcellular defence against so many different stress-induced damage that the term

‘heat shock response’ has been replaced by the more general term ‘stress response’,and the associated products are now referred to as stress proteins [22, 23] Manystress proteins are also expressed in normal cells with the same function, such ascontrol of protein synthesis, folding, and translocation into organelles [24] Andafter cells have been exposed to a stress, these proteins are required to recogniseunfolded proteins and either target them for removal, prevent their aggregation orassist in their refolding into their native, functional state Five molecular chape-rones represent the minimal stress proteome: DnaK/HSP70, DnaJ/HSP40, GrpE,HSP60, and peptidyl-prolyl isomerase (cylophilin) The proteins involved in cellu-lar stress responses are the most highly conserved of all organisms [10] In biology,chaperones are specific proteins that have the function of assisting other proteins

in achieving proper folding They were discovered as heat shock proteins, that is,proteins expressed in heat shock conditions The reason for this behaviour is thatprotein folding is severely affected by heat, and chaperones therefore act to coun-teract the potential damage Although most proteins can fold in the absence ofchaperones, for a minority their presence is an absolute requirement

Recent analysis has revealed that stress, rather than simply imposing tive forces, leads to subtle changes in macromolecular structures, which result in aredirection of the cell energy to allow the synthesis of heat shock proteins, whichthemselves function in restoring homeostasis [25]

destruc-Cells that produce high levels of stress proteins are better able to survive thestress damage than cells that do not [26]

The major inducible heat shock protein is HSP70 The binding activity of HSP70itself is involved in the regulation of apoptosis, where it may associate withpro-apoptotic proteins, thereby keeping these proteins in the inactive state, or play

a part in the proteasome-mediated degradation of apoptosis-regulatory proteins[27] However after a severe stress, when repair turns out to be impossible HSP 70

is involved in activation of the apoptotic programme and, in the extreme case, ofcellular necrosis [28]

Oxidative stress

Oxidative stress is the cumulative production of ‘reactive oxygen species’ (ROS)and ‘reactive nitrogen species’ (RNS) through either endogenous or exogenousinsults Most endogenously formed ROS pass into mitochondria through a leakfrom a respiratory electron, resulting in the formation of superoxide anion radicals.Eventually these anion radicals are transformed into hydrogen peroxide and theninto hydroxyl radicals, HO, which directly attack surrounding macromolecules,including lipids, proteins and DNA [29] Most of this damage cannot be entirelyrepaired or removed by elements of the cellular degradative system, such asproteasomes, lysosome, cytosolic and mitochondrial proteases Consequently,irreversibly damaged and defective structures accumulate within long-lived post-mitotic cells, such as cardiac myocytes [30] and neurones [31], which explains whyage-related changes occur in any aerobic organism, especially within long-livedpostmitotic cells, even in an absolutely favourable environmental condition, lead-ing to a progressively high probability of death [32] It is also common in manytypes of cancer cell that are linked with altered redox regulation of cellular signall-ing pathways; the redox imbalance may consequently be related to oncogenicstimulation DNA mutation is a critical step in carcinogenesis, and high levels ofoxidative DNA lesions have been noted in diverse tumours, strongly implicatingsuch damage in the aetiology of cancer It appears that the DNA damage is linkedpredominantly with the initiation process [33]

Numerous stress response mechanisms are rapidly activated in response tooxidative insults Some of the pathways are preferentially linked to enhancedsurvival, while others are more frequently associated with cell death All cells havefree radical scavenging systems to diminish and repair oxidative damage, and theseinclude compounds such as glutathione, ascorbate, thioredoxin and various antio-xidant enzymes [34]

Osmotic stress

The cellular response to osmotic stress ensures that the concentration of waterinside the cell is maintained within a range that is compatible with biologicalfunction Mammals limit osmotic stress by establishing an internal aqueous envi-ronment in which intravascular water and plasma electrolyte concentrations aresubject to sensitive and dynamic, organism-based homeostatic regulation by thekidney, resulting in a homeostatic balance in which plasma osmolality does notnormally vary by more than 2–3% [35] During osmotic stress total osmolyteconcentrations can vary by hundreds of millimoles

Cells respond to osmotic stress by varying the concentration of osmolyteswithin the cell, in this manner eliminating any change in intracellular waterconcentration and the associated change in cell volume that might occur byosmosis A direct cellular response to hypertonic stress takes place in seconds andinvolves increases in the intracellular concentrations of charged ions, such as

sodium, potassium and chloride, which are mediated by pre-existing ion transportsystems [36–38].

Mammalian inner renal medullary cells are normally exposed to extremely highNaCl concentrations This condition promotes DNA damage and inhibition ofDNA repair Under normal conditions, most cells in the body die when exposed tohigh NaCl, but these renal cells mostly survive and keep functioning both in vitroand in vivo [39] The interstitial NaCl concentration in parts of a normal renalmedulla can be 500 mM or more, depending on the species [40] Several studieshave shown protective adaptations for cellular survival and functioning in thisextreme stress condition, including accumulation of large amounts of organicosmolytes, which regulate cell volume and intracellular ionic strength despite thehypertonicity of the high NaCl [41]

Endoplasmic reticulum stress

Correct functioning of the endoplasmic reticulum (ER) is essential for numerousaspects of cell physiology, including lipid and membrane biogenesis, vesicle traf-ficking and protein targeting and secretion The ER is highly susceptible to altera-tions in homeostasis and exerts a strict quality control system to ensure that onlycorrectly folded proteins transit to the Golgi Unfolded or misfolded proteins areretained in the ER and conserved cell stress response The aim of this, initially, was

to compensate for the damage, but it can eventually promote cell death if ERdysfunction is severe or prolonged [43] ER-initiated cell death is linked withseveral diseases, including hypoxia, ischaemia/reperfusion injury, neurodegenera-tion, heart disease, viral infection and diabetes, and it reflects an extreme condition

Under chronic ER stress, inositol requiring-1 (IRE1), an ER-resident brane protein kinase, is activated, leading to the recruitment of JIK (c-Jun-N-ter-minal-inhibitory kinase), and TNF-receptor-associated factor 2 (TRAF2) TRAF2activates c-Jun N-terminal protein kinase (JNK) and downstream proapoptotickinases, such as apoptosis-signalling kinase 1 (ASK1), finally directing the activa-tion of mitochondrial apoptotic protease-activating factor-1 (Apaf-1)-dependentcaspase [46] The mechanism underlying apoptosis via IRE1-JNK signalling has notyet been identified On the other hand, the recruitment of JIK enables the activation

transmem-of procaspase-12 located in the ER Once activated, caspase-12 activates pase-9 to activate procaspase-3, the executioner of cell death [47]

procas-Like IRE1, PKR-like ER kinase (PERK) is another sensor of reticulum stress.Activated PERK phosphorylates eukaryotic translation initiation factor-2 (eIF2a),which enhances translation of activating transcription factor-4 (ATF4) mRNA

ATF4 induces transcription of the pro-apoptotic factor CHOP, a member of theC/EBP family of transcription factors It has recently been shown that CHOPsensitises cells to ER stress transcriptionally, down-regulating the anti-apoptoticprotein Bcl-2 [48].

Ischaemia/hypoxia

Cellular hypoxia occurs in various conditions, ranging from environmental sures such as ascent to a high altitude to pathophysiological states with inadequateoxygen supply (hypoxia), which are usually caused by blood vessel constriction orobstruction (ischaemia) The basis of this disorder is the exhaustion of energysupplies Therefore, human cells have evolved an ability to survive and adapt toreduction of oxygen pressure in the ambience [49] Functionally, these adaptationsinclude compensatory changes that allow cells to survive the hypoxic exposureitself, such as increases in anaerobic metabolism and initiation of a cell stressresponse, in addition to responses that are designed to increase local oxygendelivery, such as production of angiogenic factors and erythropoietin [50, 51].Changes in gene expression have already been linked with the human cellularresponse to hypoxia [52] At least three important mechanisms for altering geneexpression during hypoxia have been identified: (1) changes in transcription me-diated by well-described transcription factors, including hypoxia-inducible factor-(HIF-1); (2) stabilisation of hypoxia-sensitive RNA species, such as vascular endo-thelial growth factor (VEGF); and (3) translation through the internal ribosomalentry sites (IRES), which happens in a cap-independent manner of molecules such

expo-as VEGF even under severely hypoxic conditions [53]

HIF-1 is a transcription factor consisting of a- and b-subunits HIF-1a sion is linked to cellular oxygen status, whereas the HIF-1b subunit is constitutivelyexpressed HIF-1a dimerises with HIF-1b in the nucleus and transcriptionallyactivates a number of genes by way of binding to hypoxia-responsive elements(HREs) The HIF-1a subunit is stabilised during hypoxia, but degrades rapidly viathe ubiquitin pathway in normoxia HIF-1 induces expression of proteins thatmight assist cell survival during hypoxia, such as VEGF [54]

expres-In mammals hypoxia has been well documented, and this stressful situationelicits other stress conditions by the reduction of four different parameters: (a)body temperature, (b) heart rate, (c) respiratory rate and (d) blood pH Thesedecreases are associated with a protective physiological effect; however, a longperiod of hypoxia/ischaemia causes extensive damage [55, 56]

Stress-activated signalling cascades

Many distinct steps in the stress initiation process are widely regulated by lar modifications, and particularly phosphorylation The stress-activated signall-ing cascades in stressed cells are becoming clear At the beginning of these signall-

molecu-ing cascades are the sensors of environmental stress: a family of serine/threoninekinases This family includes: PKR, RNA-dependent protein kinase, which is acti-vated by viral infection, ER stress, hypoxic stress, heat and UV irradiation [57, 58];PERK (RNA-dependent protein kinase-like endoplasmic reticulum kinase)/PEK(pancreatic eIF2alpha kinase), resident ER proteins, are activated with the accu-mulation of unfolded proteins in the ER [59]; MAPKs p38, ERK and JNK arestress-responsive and are activated by oxidative stress, such as an increase incellular H2O2[60].

The end-points of signalling events include both quick responses, such asprotein modifications, and slow responses, including transcriptional regulation,cell-cycle control, cell proliferation and cell death [13]

The endpoint of cell shock

Programmed cell death

The term ‘programmed cell death’ (PCD) was created to describe a physiologicalprocess that eliminates unwanted cells [61, 62], an active and controlled process ofself-destruction [63] Glucksmann was one of the scientists who discovered in 1951that PCD was an integral part of normal embryonic development [64]

PCD can be defined as a sequence of biochemical and morphological alterationsbased on cellular metabolism and leading to cell demise, by which dying cells areremoved in a safe, noninflammatory manner In physiological conditions, PCD istightly controlled and regulates the balance between proliferation and differentia-tion both in the course of development and during the optimisation of adult celland tissue functions, in accordance with environmental stimulus Alterations inthe regulation of PCD have been implicated in a number of pathologies, includingneurodegeneration and cancer [65–67]

PCD can be divided into four different types: apoptotic cell death, autophagiccell death, apoptosis-like PCD and necrosis-like PCD What the various types ofPCD have in common is that they are executed by active cellular processes and can

be interrupted by interfering with intracellular signalling [68]

Apoptotic cell death or type I PCD The main physical and biochemical

hall-marks of apoptosis include loss of sialic acid, translocation of phosphatidylserine

to the outer plasma membrane, cell shrinkage, nuclear condensation, chromatinaggregation, DNA fragmentation, membrane blebbing and formation of apoptoticbodies Certain modifications that occur in the plasma membrane enable therecognition of apoptotic bodies by neighbouring cells or phagocytes, preventing

an inflammatory reaction [69, 70] Apoptosis can be considered a mild response

of cells when stress exceeds cellular tolerance limits

Apoptosis consists of at least two phases: initiation and execution This totic cascade can be initiated via two major pathways in mammalian cells: theextrinsic or death receptor pathway and intrinsic or mitochondrial pathways Upon

apop-triggering of either pathway, a specific family of cysteine proteases, the caspases,

is activated to execute the programme We have to keep in mind the significantcross-talk and feedback between the different pathways that regulate the apoptoticmachinery and can promote amplification of the apoptotic stimulus [71]

The extrinsic apoptosis pathway is induced upon the binding of ligands (TNF,TRAIL, FasL etc.) to members of the TNFa receptor super-family, which are usuallycalled the death receptors (Fas, also called CD95/Apo-1; TNF receptors; TRAILreceptors) Death receptors contain an intracellular globular interaction domainknown as a death domain (DD) in the cytoplasm tail Ligand-induced receptormultimerisation results in the formation of the death-inducing signalling complex(DISC) that includes the death receptor, intracellular adaptor proteins (TRADD,FADD, RAIDD) and initiator caspases (procaspase 8), leading to autocatalyticprocessing and activation of the initiator, caspase 9 [72]

The intrinsic pathway is initiated by the majority of apoptotic stimuli, including

UV radiation, gamma irradiation, heat, DNA damage, the actions of some proteins and tumour suppressor genes, viral virulence factors and most chemo-therapeutic agents, irradiation, cytotoxic drugs, granzyme B and DNA damage.These stimuli lead to the loss of mitochondrial membrane potential, with therelease of pro-apoptotic cell death proteins resulting in the formation of anothermultiprotein complex, the apoptosome, that includes Apaf-1, cytochrome-c,ATP/dATP and the initiator caspase, procaspase 9, promoting the autocatalyticactivation of caspase-9 and subsequent effector caspases Pro- and anti-apoptoticproteins of the Bcl-2 family regulate the release of pro-cell death mitochondrialproteins, while the activity of caspases is negatively regulated by the IAPs Smacand Omi promote caspase activation by antagonising the inhibitory effects of theIAPs, while AIF and EndoG contribute to caspase-independent cell death [73].The typical pathways of caspase activation during initiation include the ‘death-receptor-mediated’ recruitment of procaspase-2, procaspase-8 and procaspase-10and a ‘mitochondrial’ pathway through which caspase-9 is activated via release ofcytochrome-c The two pathways converge, leading to activation of procaspase-3and, further downstream, to activation of caspase-6 and caspase-7 All thesepathways are associated with activation of caspase-activated DNase (CAD), and soalso with ‘typical’ internucleosomal DNA fragmentation [74]

onco-Autophagic cell death or type II PCD Autophagy is characterised by the

accu-mulation of autophagic vesicles (autophagosomes and autophagolysosomes) anddepends on autophagy proteins It is often observed when massive cell elimination

is demanded or when phagocytes do not have easy access to the dying cells The set

of proteins (Atg5, Atg6, and Atg7) and the arrangement of autophagosomes volved in both autophagic cell death and autophagy that promotes cell survival arethe same, but their regulation is substantially different during each of these processes[75] The activation of autophagic cell death is common during tissue remodellingprocesses, such as metamorphosis in insects and organ morphogenesis duringdevelopment, and is part of the cellular response to oxidative stress [76, 77].Suppressing autophagosome formation by means of autophagy inhibitors, such as

in-3-methyladenine (3-MA) and wortmannin, or by silencing Atg5 and Atg6 inhibitsthis nonapoptotic form of cell death These results suggest that autophagosomeformation is required for cells to die after exposure to different cell stressors,proving the existence of this alternative death mechanism [78] Investigation ofautophagic death is still in its early stages, which is why information on themolecular basis of autophagic death is extremely limited.

Apoptosis-like, or type III, PCD Apoptosis-like PCD involves

caspase-inde-pendent mitochondrial pathways Upon mitochondrial outer-membrane bilisation, AIF is released from the inter-membrane mitochondrial space AIF isthe best-characterised caspase-independent cell death regulator, and upon release

permea-it translocates to the nucleus, where permea-it is associated wpermea-ith large-scale DNA tation; however, chromatin condensation is less compact than in apoptosis [79].The DNA-degrading capacity of AIF relies on recruitment of downstream nuclea-ses, such as cyclophilin A [80], and the display of phagocytosis-recognition mole-cules occurs before lysis of the plasma membrane [68]

fragmen-Necrosis-like, or type IV, PCD In necrosis-like PCD, the cell-death programme

is triggered by organelles other than mitochondria, such as ER, lysosomes, and thenucleus, and by proteases other than caspases, such as calpains and cathepsinsoriginating from the ER and lysosomes, respectively No chromatin condensation

is observed The molecular mechanisms of such PCD are less well understood,although it is believed that they represent ‘alternative’ death pathways whencaspases are inhibited Ca2+and ROS can lead on to severe mitochondrial dysfunc-tion and necrosis-like PCD with or without autophagy [81]

Both apoptosis and necrosis-like PCD are induced by chemotherapy, whichcauses cellular stress [82]

Necrosis

Necrosis is a more disorderly manner of cell death, which results from harshcircumstances outside the cell and is often called ‘accidental’ cell death, since itusually occurs as a result of unintentional traumatic injury, whether thermal,chemical or anoxic It is characterised by DNA broken into randomly sized frag-ments, cellular oedema and disruption of the plasma membrane, leading to release

of the cellular components and inflammatory tissue response [83] Phosphatidilserine externalisation, an event previously considered unique for apoptosis, mayalso occur in cells undergoing necrosis [84]

Necrosis has a major role in neuronal cell death following neonatal xia/ischaemia Cytochrome-c release and caspase activation were also noted invarious human breast carcinoma cells induced by a cytotoxic agent to undergonecrosis [83]

hypo-Apoptosis and necrosis have been shown to be more similar in their regulationthan was previously believed, with several signalling pathways in common There

is often a delicate balance between the two modes of death, yet outcomes andconsequences for the organism can be totally different, depending on whichpathway is followed after a cell stress.

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phospha-Tissue partial pressure of carbon dioxide tension

measurements and microcirculation visualisation New techniques for the study of low flow states

G RISTAGNO, W TANG, M.H WEIL

Microcirculation is the ultimate determinant of the outcomes of circulatory shockstates Microcirculatory function is the prerequisite for adequate tissue oxygena-tion and therefore organ function It transports oxygen and nutrients to tissue cells,ensures adequate immunological function and, during disease, delivers therapeuticdrugs to target cells It is made up of the smallest blood vessels: arterioles, capillariesand venules [1] (Fig 1) The previous techniques used for studying microcircula-tion (microscopes, laser Doppler or plethysmography) were able to provide only aglobal measurement of microvascular blood flow; a measurement expressed as anaverage value of whatever was the diameter or direction of single vessels Recenttechnological developments allow more precise and direct investigation of thetissue perfusion, and especially of the microcirculatory blood flow The new tech-niques are basically noninvasive measurements of tissue carbon dioxide tension

(PCO2), for example at the oral cavity mucosa, and the orthogonal polarisationspectral (OPS) imaging techniques, which have allowed direct visualisation andmonitoring of microcirculation at the bedside [2, 3] (Fig 2)

Fig 1 Anatomy of microcirculation

Tissue CO2measurements

Tissue hypercarbia accompanies diverse states of perfusion failure, and it is nised as a diffuse phenomenon during circulatory shock It has in fact beenobserved in heart, stomach, liver, kidney and brain in conditions of haemorrhagic

recog-and anaphylactic shock [4–9] Increases in tissue PCO2 account for anaerobicproduction of CO2 In fact, when oxygen delivery to the tissues is critically reduced,during circulatory failure states, anaerobic metabolism is triggered with conse-quent hydrogen ion production This excess of hydrogen ions is buffered by tissuebicarbonate in such a way that CO2is generated [9, 10] In the first measurements

of tissue PCO2, gastric tonometry was recognised as an early and clinically usefulindicator of perfusion failure during low flow states [11] Gastric tonometry isaccomplished by way of a balloon incorporated in the distal end of a nasogastrictube, which is advanced into the stomach The balloon is then filled with saline

solution, and after an interval of 45–90 min of equilibration, the PCO2of the fluidsampled from the balloon is measured with a conventional blood gas analyser Thistechnique also provides for analyses of the gastric intramucosal pH (pHi), which

is computed from simultaneous measurements of the PCO2 in the saline and

calculation of bicarbonate from arterial blood measurements of pH and PCO2based on the Henderson-Hasselbach equation Several clinical studies have con-firmed the validity and the utility of gastric tonometry Close correlations betweengastric pHi and mortality have been reported [12] In 83 patients with acutecirculatory failure, gastric pHi measured by tonometry was compared with ade-quacy of tissue oxygenation assessed by conventional methods (cardiac index,oxygen delivery and oxygen uptake) Only gastric pHi at 24 h proved to be an

Fig 2 Orthogonal Polarization Spectral imaging camera: CYTOSCAN A/R (Cytometrics Inc.,Philadelphia, PA)

independent predictor of outcome, predicting death with a sensitivity of 88% [13].However, the tonometric method presented several limitations [14] It was an

invasive method, which required stopping feeding The tissue PCO2measurements

could be influenced by the PCO2of the gastric juice and by the PCO2generated inthe gastric wall as a result of the neutralisation of hydrogen ions by the bicarbonatecontained in the gastric juice or in the backflow of duodenal fluid Therefore, thismeasurement required H2-blockade Gastric tonometry also presented relativelylabour-intensive manipulations and a long time interval for equilibration of CO2between the saline in the tonometer balloon and the gastric wall For all thesereasons and also because we recognised that hypercarbia was a general phenome-non in perfusion failure, which was equally profound in the intraabdominal visceraand in extraabdominal sites in circulatory shock, we investigated diverse sites for

measurement of tissue PCO2directly and in less invasive ways [6]

We had previously demonstrated a close correlation between gastric and

oeso-phageal wall mucosa PCO2[5], and subsequently we established that sublingualfossa mucosa and buccal mucosa were sites that provided measurements of tissue

PCO2comparable to those in the mucosa of both the stomach and the oesophagealwall In fact, decreases in organ blood flow were closely associated with increases

in PCO2in the sublingual mucosa and that of the buccal cavity [5–7, 15–17] We also

investigated the feasibility and predictive value of sublingual PCO2(PslCO2) urement as a noninvasive and early indicator of systemic perfusion failure on

meas-clinical settings PslCO2was measured in five healthy human volunteers and in 46patients with acute illness or injuries admitted to ICUs attached to emergency

departments and to medical and surgical departments PslCO2was approximately

45 torr in the healthy volunteers and approximately 81 torr in 26 patients who

presented signs of circulatory failure The initial sublingual mucosa PCO2of 12patients who died without recovering from shock was approximately 93 torr, and

this contrasted with the value of 58 torr (p<0.001) in hospital survivors When PslCO2exceeded the threshold of 70 torr its positive predictive value for presence

of physical signs of circulatory shock was 1.00 A value < 70 torr predicted survivalwith a predictive value of 0.93 Later, we demonstrated that the buccal mucosa

tissue PCO2measurements could be used as sensitive indicators of systemic bloodflow during haemorrhagic shock [16] We induced haemorrhagic shock in fiveanaesthetised pigs weighing 35–40 kg Blood was shed at a rate of 20 ml/min untilthe mean arterial pressure had declined to 55±5 mmHg After 2 h the shed bloodwas reinfused at a rate of 100 ml/min and animals were observed for a further 2 h

Over the 2-h interval of shock, the buccal mucosa PCO2increased in parallel with

the sublingual mucosa PCO2, from 56 to 116 torr (Fig 3) Increases in buccal tissue

PCO2were accompanied by corresponding decreases in cardiac output and meanarterial pressure, and by increases in arterial blood lactate concentrations In-

creases in buccal PCO2were accompanied by reductions in buccal mucosal flows,measured by microsphere techniques These decreases in blood flow were closelyrelated to those in the sublingual sites and to concomitant reductions in liver andkidney blood flow After reinfusion of the shed blood, buccal and sublingual

mucosa PCO2 values were restored to baseline There was a close correlation