The role of molecular diffusion within dendritic spines in synaptic function

Spines are tiny nanoscale protrusions from dendrites of neurons. In the cortex and hippocampus, most of the excitatory postsynaptic sites reside in spines. The bulbous spine head is connected to the dendritic shaft by a thin membranous neck. Because the neck is narrow, spine heads are thought to function as biochemically independent signaling compartments. Thus, dynamic changes in the composition, distribution, mobility, conformations, and signaling properties of molecules contained within spines can account for much of the molecular basis of postsynaptic function and regulation. A major factor in controlling these changes is the diffusional properties of proteins within this small compartment. Advances in measurement techniques using fluorescence microscopy now make it possible to measure molecular diffusion within single dendritic spines directly. Here, we review the regulatory mechanisms of diffusion in spines by local intra-spine architecture and discuss their implications for neuronal signaling and synaptic plasticity.

IntroductionNeurons communicate with each other through synapses that organize to create functional circuits. Most excitatory synapses in the central nervous system are formed on dendritic spines, tiny protrusions that extend from dendrites (Bourne and Harris, 2008; Fig. 1 a). The spine typically has a head of 200–1,000-nm diameter, which is connected to the dendritic shaft via a neck of 100–200-nm width (Arellano et al., 2007; Fig. 1 b). The head contains postsynaptic density (PSD) proteins, the actin cytoskeleton, membrane structures, and organelles (Sheng and Hoogenraad, 2007; Fig. 1 c). The molecular composition of spine heads is different from that of the shaft. Because of its characteristic morphology, spines are thought to function as biochemically independent compartments by limiting molecular movement between the spine head and the rest of the dendrite (Adrian et al., 2014; Tønnesen and Nägerl, 2016). Clarifying this regulation is key to understanding how this unitary site of synaptic transmission is controlled. This is particularly crucial to our understanding about how changes in the postsynaptic site lead to synaptic plasticity.Open in a separate windowFigure 1.The shape and internal architecture of dendritic spines. (a) A super-resolution SIM image of a hippocampal neuron dendrite expressing GFP. (b) A surface image of a spine (arrow in a) reconstructed from a SIM image. (c) Schematic representation of a spine containing the PSD, actin cytoskeleton, recycling endosome, and SER.The control of spine architecture is critical at excitatory synapses in the brain (Alvarez and Sabatini, 2007; Forrest et al., 2018). Excitatory synapses exhibit synaptic plasticity, which changes the strength of synaptic transmission through mechanisms at both pre- and postsynaptic sides (Citri and Malenka, 2008). This process is generally thought to be a basis for changes in neural circuits controlled by experiences—i.e., learning and memory (Humeau and Choquet, 2019; Magee and Grienberger, 2020). Here, the size and shape of spines are strongly correlated with the strength of synaptic transmission (Kasai et al., 2010). Spine volume is proportional to PSD area (Harris and Stevens, 1989) and the number of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate–type glutamate receptors (AMPARs; Nusser et al., 1998; Matsuzaki et al., 2001). Recently, combinational analysis of electrophysiology and correlative light and EM (CLEM) revealed the linear relationship between PSD area and synaptic strength (Holler et al., 2021). Also, longer spine necks attenuate somatic potentials to a greater degree (Araya et al., 2006). Thus, structural and functional plasticity of spines is tightly regulated. Specifically, when synaptic transmission is strengthened (e.g., long-term potentiation [LTP]), spines grow (Matsuzaki et al., 2004). In turn, when synaptic transmission weakens (e.g., long-term depression), spines shrink (Zhou et al., 2004; Oh et al., 2013).While many molecules involved in the plasticity of spine synapses have been identified (Sala and Segal, 2014), their mechanisms and regulations can only be discovered by monitoring the regulated changes in the composition and signaling properties of these factors within the confined space of the spine’s cytoplasm. To this end, the development of local photolysis of caged-glutamate played an important role (Matsuzaki et al., 2001). This method made it possible to induce structural plasticity locally at a single spine. Spine enlargement is induced by uncaging of caged-glutamate in the absence of Mg²⁺ or with postsynaptic depolarization in the presence of Mg²⁺ to activate N-methyl-D-aspartate–type glutamate receptors (NMDARs; Matsuzaki et al., 2004). Conversely, spine shrinkage is induced by low-frequency uncaging of caged-glutamate in the absence of Mg²⁺ or with postsynaptic depolarization (Oh et al., 2013). Shrinkage can also be induced by glutamate uncaging temporally coupled with back propagation action potential and uncaging of caged–γ-aminobutyric acid (GABA; Hayama et al., 2013). Glutamate uncaging–induced structural plasticity has also been seen to occur in vivo (Noguchi et al., 2019).These methods have vastly improved our understanding of the molecular mechanisms of structural plasticity (Nishiyama and Yasuda, 2015). In particular, stimulus-dependent increases in spine size (structural LTP [sLTP]), which are thought to be associated with functional LTP, have been studied extensively as a model of LTP (Nakahata and Yasuda, 2018; Fig. 2). Strong synaptic input causes an influx of Ca²⁺ through NMDARs that activates Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the downstream signaling cascades. This modification of signaling cascades can affect cytoskeletal organization and membrane trafficking, which are responsible for two subsequent cellular events. First, spine morphology is modulated through cytoskeletal changes (Borovac et al., 2018). Second, synaptic transmission is enhanced by increased AMPAR insertion into the plasma membrane and movement to the PSD (Huganir and Nicoll, 2013). However, the molecular mechanisms that link these two phenomena are not fully understood (Herring and Nicoll, 2016). As sLTP progresses, the molecular composition within spines changes (Bosch et al., 2014; Meyer et al., 2014). Specifically, immediately after sLTP induction, actin-related molecules such as cofilin and actin-related protein 2/3 (Arp2/3) complex accumulate within a stimulated spine. On the other hand, scaffold proteins such as PSD-95 slowly accumulate over tens of minutes. SynGAP, which is localized at the PSD through interaction with PSD-95, escapes from spines immediately after stimulation and contributes to the expression of sLTP (Araki et al., 2015). These changes in molecular compositions immediately after stimulation may be due not only to molecule-specific binding but also to the physical regulation of diffusion (Obashi et al., 2019).Open in a separate windowFigure 2.Molecular motion important in sLTP. Strong synaptic input causes an influx of Ca²⁺ through NMDARs that activates CaMKII and the downstream signaling cascades. This modification of signaling cascades can affect cytoskeletal organization and membrane trafficking, which regulate spine morphology. Spine morphology affects the molecular exchange between the spine head and the dendritic shaft and lateral diffusion of membrane proteins including AMPARs. Regulation of molecular movements through the spine neck affects the molecular composition within spines. This change affects signal propagation into nearby spines. For example, cofilin and Arp2/3 complex accumulate within spines. SynGAP and activated RhoA escape from spines. Reorganization of the actin cytoskeleton affects movement of large molecules and the formation of a large signaling complex containing CaMKII and Tiam1. Also, the structure of the PSD affects membrane protein diffusion and alters the synaptic trafficking of AMPARs.In addition to molecular localization, fluorescence lifetime imaging of FRET-based biosensors has made it possible to measure spatiotemporal changes in the activity of signaling molecules involved in sLTP (Yasuda, 2012). These studies have demonstrated a critical relationship between the time that a molecule spends within a spine and the rate of signal inactivation. This relationship determines whether an activated signaling molecule is confined within a single spine or escapes from the spine and interacts with effectors present in the adjacent dendritic shaft or nearby spines (Yasuda, 2017). The signal propagation into nearby spines is most likely related to heterosynaptic plasticity, where activated synapses influence neighbor synapses within the same dendritic segments (Oh et al., 2015; Colgan et al., 2018; Chater and Goda, 2021). Thus, diffusion is a central feature of the regulation of spine structural plasticity. However, because the size of spines is small relative to the spatial resolution of diffraction-limited fluorescence microscopy and measuring methods are limited, elucidation of the mechanism regulating diffusion within spines has been challenging.To address this gap in understanding, researchers advanced fluorescence microscopy techniques, which enabled us to measure changes in the nanoscale localization, diffusion, and signal activities inside spines. These studies allow us to directly understand how spine structures physically limit molecular diffusion and reveal fundamental mechanisms that control the localization and biochemical signal transduction pathways in neurons. Here, we summarize recent findings that have revealed physical barriers within spines using super-resolution microscopy (Sigal et al., 2018; Table 1) and molecular dynamics measurements (Fig. 3 and Table 2), and we discuss how these barriers serve as a fundamental feature controlling neuronal signaling and synaptic plasticity.Table 1.List of super-resolution microscopy techniques

A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.     

Comparison of Insulins Glargine and Degludec in Diabetic Rhesus Macaques (Macaca mulatta) with CGM Devices

Treating and monitoring type 2 diabetes mellitus (T2DM) in NHP can be challenging. Multiple insulin and hypoglycemic therapies and management tools exist, but few studies demonstrate their benefits in a NHP clinical setting. The insulins glargine and degludec are long-acting insulins; their duration of action in humans exceeds 24 and 42 h, respectively. In the first of this study''s 2 components, we evaluated whether insulin degludec could be dosed daily at equivalent units to glargine to achieve comparable blood glucose (BG) reduction in diabetic rhesus macaques (Macaca mulatta) with continuous glucose monitoring (CGM) devices. The second component assessed the accuracy of CGM devices in rhesus macaques by comparing time-stamped CGM interstitial glucose values, glucometer BG readings, and BG levels measured by using an automated clinical chemistry analyzer from samples that were collected at the beginning and end of each CGM device placement. The CGM devices collected a total of 21,637 glucose data points from 6 diabetic rhesus macaques that received glargine followed by degludec every 24 h for 1 wk each. Ultimately, glucose values averaged 29 mg/dL higher with degludec than with glargine. Glucose values were comparable between the CGM device, glucometer, and chemistry analyzer, thus validating that CGM devices as reliable for measuring BG levels in rhesus macaques. Although glargine was superior to degludec when given at the same dose (units/day), both are safe and effective treatment options. Glucose values from CGM, glucometers, and chemistry analyzers provided results that were analogous to BG values in rhesus macaques. Our report further highlights critical clinical aspects of using glargine as compared with degludec in NHP and the benefits of using CGM devices in macaques.

Diabetes is a group of metabolic diseases that cause hyperglycemia secondary to deficient insulin response, secretion, or both.⁴ Diabetes is categorized by the American Diabetes Association into 4 types: 1) type 1 diabetes mellitus, in which the pancreas is unable to produce insulin for glucose absorption; 2) type 2 diabetes mellitus (T2DM), when the body does not use insulin correctly; 3) gestational diabetes, in which the body is insulin-intolerant during pregnancy (or is first discovered then); and 4) other specific forms of diabetes in which the patient is particularly predisposed to becoming diabetic due to various comorbidities or to inadvertent induction caused by some medications.⁴ In 2018, 34.2 million (10.5%) Americans of all ages were diagnosed with diabetes.^22,23,30 Approximately 90% to 95% of Americans with diabetes have T2DM,²⁴ making T2DM the most common form of diabetes diagnosed in humans.T2DM is a multifactorial disease primarily determined by genetics, behavioral and environmental factors (for example, age, diet, sedentary lifestyle, obesity).^4,46,50,74 As a consequence of these factors, the pancreas increases insulin secretion to maintain normal glucose tolerance.⁷⁴ Over time, the high insulin demand causes pancreatic β-cell destruction, resulting in reduced production of insulin.^39,50,74 As β-cell destruction increases, hyperglycemia and T2DM develop. Insulin resistance and hyperglycemia are tolerated for a period of time^19,82,83 before clinical signs associated with T2DM develop (e.g., polydipsia, polyuria, polyphagia with concurrent weight loss).⁴ Once clinical signs develop, T2DM is most commonly diagnosed as a fasting blood glucose level (FBG) of 126 mg/dL or greater,^2,4 2-h plasma glucose value of 200 mg/d or greater during a 75-g oral glucose tolerance test,^2,4 and/or glycosylated hemoglobin (HbA1c) of 6.5% or greater.^2,4 Depending on the FBG, oral glucose tolerance test, and HbA1c results, various treatment options are recommended by the American Diabetes Association. Most importantly, lifestyle changes, including diet and exercise, are recommended as the first line of treatment, along with oral antihyperglycemic drugs such as metformin.^5,25,46 Treatment efficacy is evaluated with self-monitoring blood glucose or continuous glucose monitoring (CGM) devices.³ Human patients using CGM devices have achieved considerable reductions in HbA1c compared with patients not using them.³ As CGM devices have become more readily available, user friendly, and affordable, they have become an essential tool in managing T2DM.Similar to humans, most NHP affected by diabetes are diagnosed with T2DM.^80,83 NHP are predisposed to similar genetic, behavioral and environmental factors (e.g., age, diet, sedentary lifestyle, obesity);^{6,18,19,37,44,52,82,83} have similar pathophysiology;^38,81-83 are diagnosed via FBG,^39,83 HbA1c,^21,31,49,56 fructosamine,^20,83,87 and weight loss;^49,80,83,86 and are treated with exercise and diet modifications as a first line of treatment.^{11,19,39,53,79} Although the human and NHP conditions are similar, the treatment and management of T2DM is somewhat different, especially when NHP have restricted physical activity due to housing constraints.Previous studies indicate that daily dosing with insulin glargine achieves appropriate glycemic control in NHP.⁴⁸ Therefore, we implemented glargine, along with some diet modification, to improve glycemic control in our diabetic colony. Other noninsulin therapies, such as metformin, had been used, but compliance was low (for example, due to large pill size, unpleasant taste, etc.). However, achieving glycemic control using diet modification, insulin glargine treatment, monthly scheduled FBG, quarterly HbA1c, and regular weight monitoring was challenging in a large colony. Monthly FBG and fructosamine testing were performed due to affordability and practicality for NHP in a research setting. Given that fructosamine levels correlate with BG concentrations for the preceding 2 to 3 wk and HbA1c percentages relate to BG concentration over 1.5 to 3 mo,^49,87 HbA1C was selected over fructosamine for T2DM management in our colony. Determining which T2DM treatment and diagnostics are most effective can be difficult in large colonies of NHP. Therefore, improved treatment and management strategies would help to manage T2DM in NHP more efficiently.Insulin glargine is a long-acting insulin, with a half-life of 12 h and duration of action of 12 to 24 h in humans^40,55 and 12 h in dogs.^34,43,60 Once injected subcutaneously, insulin glargine forms a microprecipitate in the neutral pH environment, which delays and prolongs absorption in subcutaneous tissues.¹² Insulin degludec is a newer form of long-acting insulin, with a half-life of 25 h^41,63,62,77 and duration of action that exceeds 42 h in humans.^40,41,68,77 Insulin degludec forms a soluble and stable dihexamer in the pharmaceutical formulation, which includes phenol and zinc.^63,78 The phenol diffuses away, leading to the formation of a soluble depot in the form of long multihexamer chains in which zinc slowly diffuses from the end of the multihexamers, causing a gradual, continuous, and extended-release of monomers from the depot of the injection site.^63,78 Pharmacodynamic studies in humans, demonstrate that the “glucose-lowering effect” of insulin degluc⁴⁰ is evenly distributed over 24 h, allowing a more stable steady-state and improved wellbeing.⁷⁸ This approach could potentially reduce the number of hypoglycemic events and provide a less rigid daily injection schedule,⁵⁸ thus potentially making insulin degludec—compared with insulin glargine—a safer, alternative diabetes therapy.In addition to medical intervention, glycemic control is achieved through regular management and monitoring of BG. Self-monitoring blood glucose checks in humans^3,5 and glucose curves in animals¹⁰ are some of the management tools used to determine or evaluate therapy for T2DM patients. Telemetry systems like CGM devices are used to monitor interstitial glucose and have been used extensively in humans^3,17,33 and animals^{16,27,36,42,47,84,85} to monitor BG in real-time. Using CGM devices 1) reduces or eliminates the number of blood draws needed to collect FBG,⁶¹ 2) accurately assesses insulin therapy via a real-time glucose curve,^72,84,85 3) allows patients and clinicians to titrate treatment^61,73 as indicated, and 4) obtains continuous glucose data with reduced manipulation and subsequent decreased stress.^72,84,85 Therefore, CGM devices can be a safe and informative tool in monitoring spontaneous T2DM in NHP.Between 2015 and 2030, the prevalence of diabetes is predicted to increase by 54% to more than 54 million Americans affected by diabetes (i.e., diabetes mellitus types 1 and 2).⁷⁰ NHP are an essential model for human T2DM because of their similar pathophysiology, diagnostics, treatment, and management. As more people develop diabetes, novel therapies will continue to be developed. Studying new treatments and management tools in NHP can further human and NHP T2DM research to prevent the progression of T2DM and hopefully diminish projections for the number of future diabetes cases. Human medical literature, American Diabetes Association, and drug manufacturers all recommend giving equal doses (i.e., number of units/day) of long-acting insulins when changing from one long-acting insulin to degludec.^26,63,67 Therefore, we hypothesized that insulin degludec would provide effective glycemic control for rhesus macaques with T2DM when dosed at equivalent doses (that is, the same number of units/day) as insulin glargine. In addition, we hypothesized that CGM devices would provide accurate BG readings as compared with chemistry analyzer and glucometer BG readings, making it a more efficient and effective tool for measurement of BG levels in rhesus macaques with T2DM.     

Inflammatory Responses with Left Ventricular Compromise after Induction of Myocardial Infarcts in Sheep (Ovis aries)

Ischemic myocardial disease is a major cause of death among humans worldwide; it results in scarring and pallor of the myocardium and triggers an inflammatory response that contributes to impaired left ventricular function. This response includes and is evidenced by the production of several inflammatory cytokines including TNFα, IL1β, IL4, IFNγ, IL10 and IL6. In the current study, myocardial infarcts were induced in 6 mo old male castrated sheep by ligation of the left circumflex obtuse marginal arteries (OM 1 and 2). MRI was used to measure parameters of left ventricular function that include EDV, ESV, EF, SVI, dp/dt max and dp/dt min at baseline and at 4 wk and 3 mo after infarct induction. We also measured serum concentrations of an array of cytokines. Postmortem histologic findings corroborate the existence of left ventricular myocardial injury and deterioration. Our data show a correlation between serum cytokine concentrations and the development of myocardial damage and left ventricular functional compromise.

Heart failure is a globally significant problem in both humans and lower animals.^3,18 The medical literature is replete with predisposing causes of heart disease,¹³ yet the prevalence of heart failure remained high.^4,5,16 Regardless of the cause of myocardial damage and subsequent left ventricular compromise, the literature indicated that the proinflammatory response that occurs after myocardial infarction is an important contributor to the deterioration of the myocardium^{1,9,12,14,17,18,20,21} Sheep and pigs are excellent translational models of human cardiology because their hearts bear many physiologic and anatomic similarities to the human heart.^4,8,15 The primary use of these models in cardiology is primarily to study myocardial infarction^5,13,16 and to a lesser extent, physiologic processes that develop after myocardial insult.Our study measured some of the major proinflammatory cytokines that contribute to myocardial damage. Most of these cytokines, including: TNFα, IL6, and IFNγ, are important correlates of myocardial ischemia that contribute to a decline in left ventricular myocardial function.^1,9,14 In our study, we detected left ventricular compromise as early as 4 wk after the infarction, while the proinflammatory response was recorded at 48 h after the infarct and peaked at 4 wk. Cardiac functional parameters began to decline early in the study consistent with the proinflammatory response. The cardiac functional parameters continued to decline until 3 mo, which was the termination of the study. These findings may support antiinflammatory intervention as an important adjunct of any therapeutic regimen.     

Effects of Buprenorphine,Chlorhexidine, and Low-level Laser Therapy on Wound Healing in Mice

Systemic buprenorphine and topical antiseptics such as chlorhexidine are frequently used in research animals to aid in pain control and to reduce infection, respectively. These therapeutics are controversial, especially when used in wound healing studies, due to conflicting data suggesting that they delay wound healing. Low-level laser therapy (LLLT) has been used to aid in wound healing without exerting the systemic effects of therapies such as buprenorphine. We conducted 2 studies to investigate the effects of these common treatment modalities on the rate of wound healing in mice. The first study used models of punch biopsy and dermal abrasion to assess whether buprenorphine HCl or 0.12% chlorhexidine delayed wound healing. The second study investigated the effects of sustained-released buprenorphine, 0.05% chlorhexidine, and LLLT on excisional wound healing. The rate of wound healing was assessed by obtaining photographs on days 0, 2, 4, 7, and 9 for the punch biopsy model in study 1, days 0, 1, 2, 4, 6, 8, 11, and 13 for the dermal abrasion model in study 1, and days 0, 3, 6, and 10 for the mice in study 2. Image J software was used to analyze the photographed wounds to determine the wound area. When comparing the wound area on the above days to the original wound area, no significant differences in healing were observed for any of the treatment groups at any time period for either study. Given the results of these studies, we believe that systemic buprenorphine, topical chlorhexidine, and LLLT can be used without impairing or delaying wound healing in mice.

A recent retrospective analysis using a medical insurance dataset estimated that approximately 8.2 million people experienced wounds ranging from acute to chronic conditions within the particular year analyzed, and estimated that the cost of acute and chronic wound treatments ranged from $28.1 to $96.8 billion dollars.⁵² The projected rise in the number of people experiencing wounds and the cost of wound care products⁵² have made wound healing a growing area of interest in both clinical medicine and research. Wound healing is a complex process that involves many overlapping, intricate physiologic processes. Each step can have associated deviations that may lead to enhanced, altered, impaired, or delayed healing. Animal research has been used to develop a better understanding of the basic, physiologic mechanisms of wound healing. Mice are the most commonly used animal in biomedical research, and they are used to model a host of conditions, including wound healing. Despite known anatomic and physiologic differences between murine and human skin,^17,53 this species is commonly used due to their small size, ease of handling, and relatively low cost. In addition, the overlapping phases of the wound healing process are similar in mice and humans, making mice a valuable model.⁶⁵Pain is inherent to the development of wound models. Pain receptors in the skin are sensitized during the actual wounding process and during the inflammatory response that occurs immediately after wounding.¹⁹ Pain can also occur during the cleansing and treatment of wounds.¹⁹ Just as managing wound pain is critical in human patients, The Guide for the Care and Use of Laboratory Animals (the Guide)³⁰ and other federal guidelines and regulations governing the care and use of laboratory animals strongly encourages the use of analgesics for animals that experience pain and/or distress.³⁰ Pain, which can also cause stress, may evoke a persistent catabolic state and may ultimately delay wound healing.^19,28,31,43 Therefore, adequate pain control is necessary to avoid negatively affecting or altering the wound healing process.As in human medicine, opioids are commonly used to provide analgesia to research rodents. Buprenorphine, a mixed agonist-antagonist opioid,^26,54 is a common analgesic that acts as a very weak partial agonist of the mu opioid receptor and an antagonist of the κ opioid receptor.²⁶ Buprenorphine is frequently used in animals as both a pre- and post-operative analgesic. It works by binding to the opioid receptors in the skin and other tissues. This ligand-receptor binding regulates the physiologic responses of nociception and inflammation,⁷ which are key factors in the process of healing and regeneration. Buprenorphine is often used instead of full mu-opioid receptor agonist drugs, such as morphine or hydromorphone, because it has fewer systemic side effects.²⁸ Despite their common use as analgesics, reports are mixed in terms of whether opioids, as a class, delay or impair wound healing.^11,28,35,40In addition to controlling pain, minimizing wound contamination and preventing infection is critical to wound healing. The use of antiseptics is often favored over the use of antibiotics as the former presents less chance for developing antibiotic resistance.⁶ As an antiseptic, chlorhexidine is commonly used to irrigate, cleanse, and treat cutaneous wounds. Chlorhexidine has high antimicrobial activity against gram-positive and gram-negative bacteria and some fungi and viruses.⁴ Although considered to be relatively safe, reports are conflicting with regard to whether chlorhexidine delays or impairs wound healing.^4,9,50,57Laser techniques have been used medically for many years, and their powerful, but precise capabilities have rendered them a unique surgical and therapeutic modality. In brief, when the electrons of atoms move to higher energy levels, these electrons absorb energy. This excited energy state is unstable and temporary. The natural return of electrons to their more stable ground state releases energy in the form of photons or light. Light Amplification by Stimulated Emission of Radiation (LASERS) are characterized by the photon stimulation of an already excited electron. This stimulation causes the emitted light to be amplified, as demonstrated by the intense, bright light that is emitted from lasers.⁶³ The concept of low-level laser therapy (LLLT) has garnered interest as a therapeutic modality in both human and veterinary medicine. Specifically characterized as laser therapy using a low power output and a low power range, LLLT is distinguished from other forms of laser therapies by certain parameters such as wavelength, pulse rate and duration, total irradiation time, and dose.⁴⁴ Although the mechanism of action for LLLT is not completely understood,^46,64 the absorption of red and near infrared light energy may reduce detrimental, inflammatory substances^13,15,24,56 while simultaneously stimulating restorative processes.^15,24,46,64 The reduced photothermal impact of LLLT⁴⁴ is reported to produce beneficial physiologic and biologic effects including analgesia, reduction in inflammation, and acceleration of healing.⁴⁸ The initial report of LLLT as a therapeutic modality found accelerated wound healing and fur regrowth in mice exposed to LLLT.^13,44,46,64 LLLT has since been used as a sole or adjunct therapy for a variety of conditions including tooth root resorption,⁵⁵ traumatic brain injuries,⁵⁸ and tendon, muscle, and bone injuries.^2,3,25,38Studies conducted to assess the effects of LLLT on healing often use parameters of normal wound healing to analyze how LLLT influences those parameters in comparison to healthy, undamaged tissue and damaged tissue not receiving laser therapy. Despite the numerous studies designed to investigate the effects of LLLT on wound healing, conflicting reports exist regarding its efficacy.^{15,17,46,22,23,24,29,34,38,39,55,56,60,64} A recent study in dogs reported accelerated healing and improved cosmetic appearance of a hemilaminectomy surgical site after LLLT,⁶⁰ while other canine studies reported no significant differences in the healing of surgically induced skin wounds between dogs that did and did not receive LLLT.^22,34 Similarly, in an attempt to study the effects of LLLT in pigs, an animal with skin very similar to that of humans, no significant differences were reported in the healing of surgically created skin wounds between swine that did and did not receive LLLT.²⁹ Studies using diabetic rats with excisional cutaneous wounds reported accelerated wound healing,^17,46 and beneficial results were reported in a similar study using diabetic mice.^56,64 While fewer studies have been conducted on the use of LLLT in rodents without concomitant comorbidities, LLLT has been reported to accelerate wound healing in healthy rodents.^15,24 Conversely, some studies found that LLLT does not accelerate or significantly improve wound healing in rodents.^24,39We performed 2 separate studies to investigate the effects of a commonly used opioid, a topical antiseptic solution, and LLLT on excisional wound healing in mice. At the time the initial study (study 1) was conducted, some of our investigators were reluctant to use the recommended analgesic, buprenorphine, due to concern about interference with their study outcomes. Therefore, we conducted study 1 to determine if a single dose of peri-operative buprenorphine would delay healing of a full-thickness excisional wound or a partial-thickness felt wheel dermal abrasion. We also examined the effects of topical chlorhexidine solution on wound healing. The chlorhexidine concentrations used in study 1 were prepared using our standard operating procedure at that time. Study 2 was conducted after study 1, with the design expanded to evaluate a sustained release buprenorphine formulation and LLLT. Study 2 used a full-thickness excisional biopsy to determine the effect of LLLT on excisional wound healing. Commonly used doses of systemic Buprenorphine Sustained Release (SR) and topical chlorhexidine were also included to evaluate their effect on excisional wound healing. The concentration of chlorhexidine in the revised, approved standard operating procedure had been decreased due to literature suggesting that higher concentrations may inhibit healing.^4,49,61 For both studies, we hypothesized that the use of buprenorphine and chlorhexidine would have no effect on the rate of wound healing, and that LLLT would accelerate wound healing in a full-thickness excision as compared with a control.     

Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague–Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 10⁶, 10⁷ or, 10⁸ CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 10⁷ or 10⁸ CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 10³ CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16.h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Protein binding is an important component of pharmacokinetic/pharmacodynamic (PK/PD) research. In vitro measurement of protein drug binding is an essential component of the research and development of novel drugs. However, in vitro studies often poorly mirror the in vivo condition.^9,42 Pharmacokinetic studies early in drug development provide a means to assess the time course of drug effects in the body and drug distribution and availability.⁴² From a PK/PD modeling perspective, protein binding is an important factor in the kinetics and dynamics of drug availability in vivo.^21,35,36,40 These complex relationships are used to project efficacious doses in humans and take into consideration differences in plasma protein binding between preclinical species and humans.^8,44A variety of acute phase proteins (APP) exist across all species and increase in response to inflammatory, infectious and traumatic events.^{5,9,12,13,19,21,22,29,45,53} APPs are potential biomarkers for detection and monitoring of various disease states including cancer.^{2,18,24,34,39,40,47,50,52} Because of this, enhanced understanding of drug binding characteristics to APPs early in the development phase will promote the design of more efficacious therapeutics. Alpha-1 acid glycoprotein (AGP), a ubiquitous major APP that is present in rats,^9,46 has significant drug binding properties and binds to many basic and neutral compounds. Normal AGP levels in plasma of naïve rats range from 0.1 to 0.32 mg/mL.⁴⁴ The importance of AGP as related to drug discovery and development will be bolstered by greater understanding of the sources of AGP stimulation in established animal models. For example, AGP modulates the immune response in a rodent shock model in which it is thought to maintain normal capillary permeability to ensure perfusion of vital organs.^30,33 In addition, elevated AGP levels are present in animal models of infection and inflammation.^{11,20,27,32,41,48}In surgically modified animals, AGP levels may be elevated after surgical manipulation, which unavoidably induces local transient inflammatory responses.^8,25,51 In addition, infections may develop postoperatively leading to increased AGP levels. Chronic catheterization has been linked to increased incidence of infection.^3,8,37 Surgically modified animals should not be placed on study if aseptic technique was not adhered to during surgical preparation and instrumentation.^6,37 Contamination may occur within or at the external portion of a catheter, usually resulting in more obvious signs of infection. Routine PK studies in rats involve implantation of vascular catheters through which drugs are administered and blood samples are taken over time. Catheterized animals are typically perceived as being healthy and thus are enrolled in and remain on study unless they develop obvious clinical signs of infection or illness. However, an occult infection may be present even with a patent catheter. As such, understanding the direct effect of subclinical infection in modulating AGP levels and drug binding is critical, as AGP levels may affect drug levels in study animals with persistent subclinical infection. In this event, the PK data generated may be altered due to selective binding to AGP, thus confounding data interpretation.A possible application of AGP is its potential utility as a biomarker for evaluating health status animals in drug development. The use of AGP as a select biomarker for monitoring and identifying sick animals and/or predicting the potential impact of subclinical infection on drug PK/PD is highly desirable. A screening tool such as this could help to optimize animal selection by reliably identifying healthy animals. Improved intra-study health monitoring would promote confidence in PK/PD data and its predictive value.The focus of this research was to develop a sensitive, reliable and reproducible model of subclinical infection in the rat using the ubiquitous skin contaminant, S. aureus. We selected AGP as a biomarker that would promote health status screening and enhance PK/PD characterization of AGP binding drugs (that is basic and neutral) in the presence or absence of subclinical infection. The model was validated by evaluating the impact of increased AGP levels on propranolol, a drug known to have high binding affinity to AGP.^{4,7,10,26,28,31,49} Ultimately, establishing this model will provide heightened visibility of the protein binding characteristics of drugs and yield more informed data interpretation.