Delirious Mania: An Approach to Diagnosis and Treatment.

The Psychiatric Consultation Service at Massachusetts General Hospital sees medical and surgical inpatients with comorbid psychiatric symptoms and conditions. During their twice-weekly rounds, Dr Stern and other members of the Consultation Service discuss diagnosis and management of hospitalized patients with complex medical or surgical problems who also demonstrate psychiatric symptoms or conditions. These discussions have given rise to rounds reports that will prove useful for clinicians practicing at the interface of medicine and psychiatry.

Longitudinal Examination of COVID-19 Public Health Measures on Mental Health for Rural Patients With Serious Mental Illness.

INTRODUCTION: There is emerging evidence to support that the COVID-19 pandemic and related public health measures may be associated with negative mental health sequelae. Rural populations in particular may fair worse because they share many unique characteristics that may put them at higher risk for adverse outcomes with the pandemic. Yet, rural populations may also be more resilient due to increased sense of community. Little is known about the impact of the pandemic on the mental health and well-being of a rural population pre- and post-pandemic, especially those with serious mental illness. MATERIAL AND METHODS: We conducted a longitudinal, mixed-methods study with assessments preceding the pandemic (between October 2019 and March 2020) and during the stay-at-home orders (between April 23, 2020, and May 4, 2020). Changes in hopelessness, suicidal ideation, connectedness, and treatment engagement were assessed using a repeated-measures ANOVA or Friedman test. RESULTS: Among 17 eligible participants, 11 people were interviewed. Overall, there were no notable changes in any symptom scale in the first 3-5 months before the pandemic or during the stay-at-home orders. The few patients who reported worse symptoms were significantly older (mean age: 71.7 years, SD: 4.0). Most patients denied disruptions to treatment, and some perceived telepsychiatry as beneficial. CONCLUSIONS: Rural patients with serious mental illness may be fairly resilient in the face of the COVID-19 pandemic when they have access to treatment and supports. Longer-term outcomes are needed in rural patients with serious mental illness to better understand the impact of the pandemic on this population.

Two new PPX2 mutations associated with resistance to PPO-inhibiting herbicides in Amaranthus palmeri.

BACKGROUND: Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO) is a widespread and growing problem for weed managers across the midwestern and midsouthern United States. In Amaranthus spp., this resistance is known to be conferred by a glycine deletion at the 210th amino acid (ΔG210) in PPO2. Preliminary analysis indicated that the ΔG210 mutation did not fully account for observed resistance to PPO inhibitors in two Amaranthus palmeri populations from Tennessee and one from Arkansas. RESULTS: Sequencing PPX2 cDNA from six resistant plants uncovered two new mutations at the R98 site (R98G and R98M), a site previously found to endow PPO-inhibitor resistance in Ambrosia artemisiifolia. Sequencing of this region from additional plants sprayed with 264 g fomesafen ha-1 showed the presence of one or both R98 mutations in a subset of the resistant plants from all three populations. No plants sensitive to fomesafen contained either mutation. A derived cleaved amplified polymorphic sequence (dCAPS) assay to test for the presence of these mutations in A. palmeri was developed. CONCLUSION: Two new mutations of PPX2 (R98G, R98M) likely confer resistance to PPO-inhibitors in A. palmeri, and can be rapidly identified using a dCAPS assay. © 2017 Society of Chemical Industry.

Resistance to aryloxyphenoxypropionate herbicides in Amazon sprangletop: Confirmation, control, and molecular basis of resistance.

Amazon sprangletop is problematic weed of rice in the midsouthern USA. Two biotypes of this species from rice fields approximately 100km apart in Louisiana were unaffected when sprayed with the labeled field rate of cyhalofop-butyl (314g ai ha-1) in 2008. Dose response studies were conducted to confirm the level of resistance to cyhalofop-butyl over a range of doses. Cross-resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides from two different chemical families and multiple herbicide resistance to other mechanisms of action were evaluated. Sequencing using the Illumina Hiseq platform and ACCase gene sequencing revealed two different amino acid substitutions, Trp2027-to-Cys in the first resistant biotype and Asp2078-to-Gly in the second resistant biotype, within the CT domain of the ACCase gene. Two known amino acid substitutions confirmed resistance to cyhalofop-butyl and fenoxaprop-P-ethyl in resistant Amazon sprangletop biotypes. Asp2078-to-Gly amino acid substitution that was detected in one of the resistant biotypes did not result in cross-resistance to clethodim, an ACCase-inhibiting cyclohexandione herbicide which has endowed clethodim resistance in other weed species. Based on this research, both resistant Amazon sprangletop biotypes have evolved target-site resistance to the APP herbicides; yet, alternative herbicides are still active on these plants.

Hemodynamics and Salt-and-Water Balance Link Sodium Storage and Vascular Dysfunction in Salt-Sensitive Subjects.

We investigated 24-hour hemodynamic changes produced by salt loading and depletion in 8 salt-sensitive (SS) and 13 salt-resistant (SR) normotensive volunteers. After salt loading, mean arterial pressure was higher in SS (96.5±2.8) than in SR (84.2±2.7 mm Hg), P<0.01, owing to higher total peripheral resistance in SS (1791±148) than in SR (1549±66 dyn*cm(-5)*s), P=0.05, whereas cardiac output was not different between groups (SS 4.5±0.3 versus SR 4.4±0.2 L/min, not significant). Following salt depletion, cardiac output was equally reduced in both groups. Total peripheral resistance increased 24±6% (P<0.001) in SR, whose mean arterial pressure remained unchanged. In contrast, total peripheral resistance did not change in SS (1±6%, not significant). Thus, their mean arterial pressure was reduced, abolishing the mean arterial pressure difference between groups. SS had higher E/e' ratios than SR in both phases of the protocol. In these 21 subjects and in 32 hypertensive patients, Na(+) balance was similar in SR and SS during salt loading or depletion. However, SR did not gain weight during salt retention (-158±250 g), whereas SS did (819±204), commensurate to iso-osmolar water retention. During salt depletion, SR lost the expected amount of weight for iso-osmolar Na(+) excretion, whereas SS lost a greater amount that failed to fully correct the fluid retention from the previous day. We conclude that SS are unable to modulate total peripheral resistance in response to salt depletion, mirroring their inability to vasodilate in response to salt loading. We suggest that differences in water balance between SS and SR indicate differences in salt-and-water storage in the interstitial compartment that may relate to vascular dysfunction in SS.

Resistance to PPO-inhibiting herbicide in Palmer amaranth from Arkansas.

BACKGROUND: The widespread occurrence of ALS inhibitor- and glyphosate-resistant Amaranthus palmeri has led to increasing use of protoporphyrinogen oxidase (PPO)-inhibiting herbicides in cotton and soybean. Studies were conducted to confirm resistance to fomesafen (a PPO inhibitor), determine the resistance frequency, examine the resistance profile to other foliar-applied herbicides and investigate the resistance mechanism of resistant plants in a population collected in 2011 (AR11-LAW B) and its progenies from two cycles of fomesafen selection (C1 and C2). RESULTS: The frequency of fomesafen-resistant plants increased from 5% in the original AR11-LAW-B to 17% in the C2 population. The amounts of fomesafen that caused 50% growth reduction were 6-, 13- and 21-fold greater in AR11-LAW-B, C1 and C2 populations, respectively, than in the sensitive ecotype. The AR11-LAW-B population was sensitive to atrazine, dicamba, glufosinate, glyphosate and mesotrione but resistant to ALS-inhibiting herbicides pyrithiobac and trifloxysulfuron. Fomesafen survivors from C1 and C2 populations tested positive for the PPO glycine 210 deletion previously reported in waterhemp (Amaranthus tuberculatus). CONCLUSION: These studies confirmed that Palmer amaranth in Arkansas has evolved resistance to foliar-applied PPO-inhibiting herbicide.

EPSPS Gene Amplification in Glyphosate-Resistant Italian Ryegrass (Lolium perenne ssp. multiflorum) Populations from Arkansas (United States).

Glyphosate-resistant Italian ryegrass was detected in Arkansas (United States) in 2007. In 2014, 45 populations were confirmed resistant in eight counties across the state. The level of resistance and resistance mechanisms in six populations were studied to assess the severity of the problem and identify alternative management approaches. Dose-response bioassays, glyphosate absorption and translocation experiments, herbicide target (EPSPS) gene sequence analysis, and gene amplification assays were conducted. The dose causing 50% growth reduction (GR50) was 7-19 times higher for the resistant population than for the susceptible standard. Uptake and translocation of (14)C-glyphosate were similar in resistant and susceptible plants, and no mutation in the EPSPS gene known to be associated with resistance to glyphosate was detected. Resistant plants contained from 11- to >100-fold more copies of the EPSPS gene than the susceptible plants, whereas the susceptible plants had only one copy of EPSPS. Plants surviving the recommended dose of glyphosate contained at least 10 copies. The EPSPS copy number was positively related to glyphosate resistance level (r = 80). Therefore, resistance to glyphosate in these populations is due to multiplication of the target site. Resistance mechanisms could be location-specific. Suppressing the mechanism for gene amplification may overcome resistance.

First report of resistance to acetolactate-synthase-inhibiting herbicides in yellow nutsedge (Cyperus esculentus): confirmation and characterization.

BACKGROUND: Yellow nutsedge is one of the most problematic sedges in Arkansas rice, requiring the frequent use of halosulfuron (sulfonylurea) for its control. In the summer of 2012, halosulfuron at 53 g ha(-1) (labeled field rate) failed to control yellow nutsedge. The level of resistance to halosulfuron was determined in the putative resistant biotype, and its cross-resistance to other acetolactate synthase (ALS) inhibitors from four different herbicide families. ALS enzyme assays and analysis of the ALS gene were used to ascertain the resistance mechanism. RESULTS: None of the resistant plants was killed by halosulfuron at a dose of 13 568 g ha(-1) (256× the field dose), indicating a high level of resistance. Based on the whole-plant bioassay, the resistant biotype was not controlled by any of the ALS-inhibiting herbicides (imazamox, imazethapyr, penoxsulam, bispyribac, pyrithiobac-sodium, bensulfuron and halosulfuron) tested at the labeled field rate. The ALS enzyme from the resistant biotype was 2540 times less responsive to halosulfuron than the susceptible biotype, and a Trp574 -to-Leu substitution was detected by ALS gene sequencing using the Illumina HiSeq. CONCLUSION: The results suggest a target-site alteration as the mechanism of resistance in yellow nutsedge, which accounts for the cross-resistance to other ALS-inhibiting herbicide families.

Physiological and molecular basis of acetolactate synthase-inhibiting herbicide resistance in barnyardgrass (Echinochloa crus-galli).

Barnyardgrass biotypes from Arkansas (AR1 and AR2) and Mississippi (MS1) have evolved cross-resistance to imazamox, imazethapyr, and penoxsulam. Additionally, AR1 and MS1 have evolved cross-resistance to bispyribac-sodium. Studies were conducted to determine if resistance to acetolactate synthase (ALS)-inhibiting herbicides in these biotypes is target-site or non-target-site based. Sequencing and analysis of a 1701 base pair ALS coding sequence revealed Ala122 to Val and Ala122 to Thr substitutions in AR1 and AR2, respectively. The imazamox concentrations required for 50% inhibition of ALS enzyme activity in vitro of AR1 and AR2 were 2.0 and 5.8 times, respectively, greater than the susceptible biotype. Absorption of ¹4C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. ¹4C-Penoxsulam translocation out of the treated leaf (≤2%) was similar among all biotypes. ¹4C-Bispyribac-treated AR1 and MS1 translocated 31- 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. ¹4C-Imazamox-treated AR1 plants translocated 39% less radioactivity above the treated leaf and aboveground tissue below the treated leaf, and MS1 translocated 54 and 18% less radioactivity to aboveground tissue above and below the treated leaf, respectively, compared to the susceptible biotype. Phosphorimaging results further corroborated the above results. This study shows that altered target site is a mechanism of resistance to imazamox in AR2 and probably in AR1. Additionally, reduced translocation, which may be a result of metabolism, could contribute to imazamox and bispyribac-sodium resistance in AR1 and MS1.

EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas.

BACKGROUND: Resistance to glyphosate in weed species is a major challenge for the sustainability of glyphosate use in crop and non-crop systems. A glyphosate-resistant Italian ryegrass population has been identified in Arkansas. This research was conducted to elucidate its resistance mechanism. RESULTS: The investigation was conducted on resistant and susceptible plants from a population in Desha County, Arkansas (Des03). The amounts of glyphosate that caused 50% overall visual injury were 7 to 13 times greater than those for susceptible plants from the same population. The EPSPS gene did not contain any point mutation that has previously been associated with resistance to glyphosate, nor were there any other mutations on the EPSPS gene unique to the Des03 resistant plants. The resistant plants had 6-fold higher basal EPSPS enzyme activities than the susceptible plants, but their I(50) values in response to glyphosate were similar. The resistant plants contained up to 25 more copies of EPSPS gene than the susceptible plants. The level of resistance to glyphosate correlated with increases in EPSPS enzyme activity and EPSPS copy number. CONCLUSION: Increased EPSPS gene amplification and EPSPS enzyme activity confer resistance to glyphosate in the Des03 population. This is the first report of EPSPS gene amplification in glyphosate-resistant Italian ryegrass. Other resistance mechanism(s) may also be involved.

Characterization of fibrodysplasia in the dog following inhibition of metalloproteinases.

Musculoskeletal side effects are a widely reported consequence of administration of particular matrix metalloproteinase inhibitors (MMPi) in clinical trials. We describe here histopathological findings during dog studies with a fairly selective MMPi AZM551248, that are consistent with these human clinical changes. They were characterized by a dose-and time-dependent formative connective tissue alteration we have termed "fibrodysplasia." The most sensitive site was the subcuticular connective tissue, although musculoskeletal tissues were also extensively involved. In the subcutis, changes occurred initially around pre-existing blood vessels, but then more diffusely. There was proliferation of cells showing myofibroblast differentiation identified by elevated levels of alpha-smooth muscle actin, fibronectin, and transforming growth factor beta, and the deposition of collagen type III with a lesser quantity of collagen type I. On longer-term administration at lower doses, there was evidence of active fibrodysplasia arising and resolving during the dosing period, resulting in the multifocal deposition of mature collagen. Although there was organ specificity, essentially identical changes occurred at multiple connective tissue sites. We conclude that MMPi-induced fibrodysplasia in animals and, by inference, musculoskeletal side effects in humans are potentially diffuse connective tissue disorders.

Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function.

Recent attempts at rebuilding the myocardium using stem cells have yielded disappointing results. The lack of a supporting vasculature may, in part, explain these disappointing findings. However, concerns over possible side effects have hampered attempts at revascularizing the infarcted myocardium using systemic delivery of proangiogenic compounds. In this study, we develop the technology to enhance the morphology and function of postinfarct neovasculature. Previously, we have shown that the up-regulated expression of endothelial cell adhesion molecules in the myocardial infarction (MI) region provides a potential avenue for selectively targeting drugs to infarcted tissue. After treatment with anti-P-selectin-conjugated liposomes containing vascular endothelial growth factor (VEGF), changes in cardiac function and vasculature post-MI were quantified in a rat MI model. Targeted delivery of VEGF to post-MI tissue resulted in significant increase in fractional shortening and improved systolic function. These functional improvements were accompanied by a 21% increase in the number of anatomical vessels and a 74% increase in the number of perfused vessels in the MI region of treated animals. No significant improvements in cardiac function were observed in untreated, systemic VEGF-treated, nontargeted liposome-treated, or blank immunoliposome-treated animals. Targeted delivery of low doses of proangiogenic compounds to post-MI tissue results in significant improvements in cardiac function and vascular structure.

A physiologically realistic in vitro model of microvascular networks.

Existing microfluidic devices, e.g. parallel plate flow chambers, do not accurately depict the geometry of microvascular networks in vivo. We have developed a synthetic microvascular network (SMN) on a polydimethalsiloxane (PDMS) chip that can serve as an in vitro model of the bifurcations, tortuosities, and cross-sectional changes found in microvascular networks in vivo. Microvascular networks from a cremaster muscle were mapped using a modified Geographical Information System, and then used to manufacture the SMNs on a PDMS chip. The networks were cultured with bovine aortic endothelial cells (BAEC), which reached confluency 3-4 days after seeding. Propidium iodide staining indicated viable and healthy cells showing normal behavior in these networks. Anti-ICAM-1 conjugated 2-mum microspheres adhered to BAEC cells activated with TNF-alpha in significantly larger numbers compared to control IgG conjugated microspheres. This preferential adhesion suggests that cultured cells retain an intact cytokine response in the SMN. This microfluidic system can provide novel insight into characterization of drug delivery particles and dynamic flow conditions in microvascular networks.

Aiming for the heart: targeted delivery of drugs to diseased cardiac tissue.

BACKGROUND: The development of a number of regenerative strategies in recent years for curing heart disease represents a paradigm shift away from conventional approaches which aim to manage heart disease. Effective administration of pharmaceutical agents targeted directly to the diseased tissue is the key to unlocking the potential of regenerative strategies, which could augment current conventional treatments. OBJECTIVE: The authors review recent advances in targeted drug delivery to diseased cardiac tissue. METHODS: Various therapeutic methodologies designed to selectively deliver pharmaceutical agents to diseased cardiac tissue are discussed in this review. CONCLUSION: Targeted delivery of survival and engraftment promoting factors to damaged cardiac tissue can be an important strategy, for example, in creating a suitable microenvironment encouraging the engraftment of stem cells. Further progress in this emerging field is contingent on the discovery of new biomarkers that are upregulated in damaged cardiac tissue and can be targeted for selective drug delivery. Once fully realized, breakthroughs in this field will have direct applications in the diagnosis and treatment of heart disease through more effective tissue-specific drug delivery and improved imaging modalities.

Synthetic microvascular networks for quantitative analysis of particle adhesion.

We have developed a methodology to study particle adhesion in the microvascular environment using microfluidic, image-derived microvascular networks on a chip accompanied by Computational Fluid Dynamics (CFD) analysis of fluid flow and particle adhesion. Microfluidic networks, obtained from digitization of in vivo microvascular topology were prototyped using soft-lithography techniques to obtain semicircular cross sectional microvascular networks in polydimethylsiloxane (PDMS). Dye perfusion studies indicated the presence of well-perfused as well as stagnant regions in a given network. Furthermore, microparticle adhesion to antibody coated networks was found to be spatially non-uniform as well. These findings were broadly corroborated in the CFD analyses. Detailed information on shear rates and particle fluxes in the entire network, obtained from the CFD models, were used to show global adhesion trends to be qualitatively consistent with current knowledge obtained using flow chambers. However, in comparison with a flow chamber, this method represents and incorporates elements of size and complex morphology of the microvasculature. Particle adhesion was found to be significantly localized near the bifurcations in comparison with the straight sections over the entire network, an effect not observable with flow chambers. In addition, the microvascular network chips are resource effective by providing data on particle adhesion over physiologically relevant shear range from even a single experiment. The microfluidic microvascular networks developed in this study can be readily used to gain fundamental insights into the processes leading to particle adhesion in the microvasculature.

Rosuvastatin: characterization of induced myopathy in the rat.

Rosuvastatin is a relatively new member of the statin family (HMG-CoA reductase inhibitors), with superior lipid-lowering effects and a pattern of clinical side effects, including a low incidence of myopathy, similar to other widely prescribed statins. This article describes investigations of myopathy in the rat following administration of very high doses of rosuvastatin. The nature of the changes were found to be entirely consistent with those seen with other statins, including a differential sensitivity of muscle fibers (with glycolytic fibers [type IIB] the most sensitive and oxidative fibers [type I] the least), a delay of approximately 10 days after the start of oral dosing before necrosis was apparent, and ultrastructural alterations appearing first in mitochondria. In addition, the development of myopathy was prevented by coadministration of mevalonate, the product of HMG-CoA reductase. The findings illustrate a pattern of induced myopathy in the rat directly attributable to inhibition of HMG-CoA reductase that is entirely consistent between the various statins, with the oral dose required to produce the changes being a differentiating feature (based on these new data and a previously reported study from the same laboratory): cerivastatin dose less than simvastatin, and simvastatin dose less than rosuvastatin.

Modeling oxygenation and selective delivery of drug carriers post-myocardial infarction.

An anatomically realistic mathematical model of oxygen transport in cardiac tissue was developed to help in deciding what angiogenic strategies should be used to rebuild the vasculature post myocardial infarction (MI). Model predictions closely match experimental measurements from a previous study, and can be used to predict distributions of oxygen concentration in normal and infarcted rat hearts. Furthermore, the model can accurately predict tissue oxygen levels in infarcted tissue treated with pro-angiogenic compounds. Immunoliposome (IL) targeting to areas of inflammation after MI could provide the means by which pro-angiogenic compounds can be selectively targeted to the infarcted region. The adhesion of model drug carriers and immunoliposomes coated with antibody to P-selectin was quantified in a MI rat model. Anti-P-selectin coated model drug carriers showed a 140% and 180% increase in adhesion in the boarder zone of the MI 1 and 4 hours post-MI, respectively. Circulating for 24 hrs, radiolabeled anti-P-selectin immunoliposomes showed an 83% and 92% increase in targeting to infarcted myocardium when injected 0 and 4 hrs post-MI, respectively. Targeting to upregulated adhesion molecules on the endothelium provides a promising strategy for selectively delivering compounds to the infarct region of the myocardium using our liposomal based drug delivery vehicle.

All-trans retinoic acid prevents development of cardiac remodeling in aortic banded rats by inhibiting the renin-angiotensin system.

This study was designed to determine the effect of all-trans retinoic acid (RA) on the development of cardiac remodeling in a pressure overload rat model. Male Sprague-Dawley rats were subjected to sham operation and the aortic constriction procedure. A subgroup of sham control and aortic constricted rats were treated with RA for 5 mo after surgery. Pressure-overloaded rats showed significantly increased interstitial and perivascular fibrosis, heart weight-to-body weight ratio, and gene expression of atrial natriuretic peptide and brain natriuretic peptide. Echocardiographic analysis showed that pressure overload induced systolic and diastolic dysfunction, as evidenced by decreased fractional shortening, ejection fraction, stroke volume, and increased E-to-E(a) ratio and isovolumic relaxation time. RA treatment prevented the above changes in cardiac structure and function and hypertrophic gene expression in pressure-overloaded rats. RA restored the ratio of Bcl-2 to Bax, inhibited cleavage of caspase-3 and -9, and prevented the decreases in the levels of SOD-1 and SOD-2. Pressure overload-induced phosphorylation of ERK1/2, JNK, and p38 was inhibited by RA, via upregulation of mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-2. The pressure overload-induced production of angiotensin II was inhibited by RA via upregulation of expression of angiotensin-converting enzyme (ACE)2 and through inhibition of the expression of cardiac and renal renin, angiotensinogen, ACE, and angiotensin type 1 receptor. Similar results were observed in cultured neonatal cardiomyocytes in response to static stretch. These results demonstrate that RA has a significant inhibitory effect on pressure overload-induced cardiac remodeling, through inhibition of the expression of renin-angiotensin system components.

Microvascular transport model predicts oxygenation changes in the infarcted heart after treatment.

Chronic heart failure is most commonly due to ischemic cardiomyopathy after a previous myocardial infarction (MI). Rebuilding lost myocardium to prevent heart failure mandates a neovasculature able to nourish new cardiomyocytes. Previously we have used a series of novel techniques to directly measure the ability of the scar neovasculature to deliver and exchange oxygen at 1-4 wk after MI in rats following left coronary artery ligation. In this study, we have developed a morphologically realistic mathematical model of oxygen transport in cardiac tissue to help in deciding what angiogenic strategies should be used to rebuild the vasculature. The model utilizes microvascular morphology of cardiac tissue based on available morphometric images and is used to simulate experimentally measured oxygen levels after MI. Model simulations of relative oxygenation match experimental measurements closely and can be used to simulate distributions of oxygen concentration in normal and infarcted rat hearts. Our findings indicate that both vascular density and vascular spatial distribution play important roles in cardiac tissue oxygenation after MI. Furthermore, the model can simulate relative changes in tissue oxygen levels in infarcted tissue treated with proangiogenic compounds such as losartan. From the minimum oxygen concentration myocytes need to maintain their normal function, we estimate that 2 wk after MI 29% of the myocardium is severely hypoxic and that the vascular density of the infarcted tissue should reach 75% of normal tissue to ensure that no areas of the myocardium are critically hypoxic.