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Hypoxia modeling techniques: A review
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Abstract
Hypoxia is the main cause and effect of a large number of diseases, including the most recent one facing the world, the coronavirus disease (COVID-19). Hypoxia is divided into short-term, long-term, and periodic, it can be the result of diseases, climate change, or living and traveling in the high mountain regions of the world. Since each type of hypoxia can be a cause and a consequence of various physiological changes, the methods for modeling these hypoxias are also different. There are many techniques for modeling hypoxia under experimental conditions. The most common animal for modeling hypoxia is a rat. Hypoxia models (hypoxia simulations) in rats are a tool to study the effect of various conditions on the oxygen supply of the body. These models can provide a necessary information to understand hypoxia and also provide effective treatment, highlighting the importance of various reactions of the body to hypoxia. The main parameters when choosing a model should be reproducibility and the goal that the scientist wants to achieve. Hypoxia in rats can be reproduced both ways exogenously and endogenously. The reason for writing this review was the aim to systematize the models of rats available in the literature in order to facilitate their selection by scientists. The relative strengths and limitations of each model need to be identified and understood in order to evaluate the information obtained from these models and extrapolate these results to humans to develop the necessary generalizations. Despite these problems, animal models have been and remain vital to understanding the mechanisms involved in the development and progression of hypoxia. The eligibility criteria for the selected studies was a comprehensive review of the methods and results obtained from the studies. This made it possible to make generalizations and give recommendations on the application of these methods. The review will assist scientists in choosing an appropriate hypoxia simulation method, as well as assist in interpreting the results obtained with these methods.
1. Introduction
A retrospective analysis of modern scientific research indicates a significant interest of the scientific community in the problems of hypoxia. The number of publications on Pubmed related to the study of hypoxia has been increasing in recent years. The dynamics of publications related to hypoxia since 1945 is shown in Fig. 1.
Interest in hypoxia can be traced in search engines. The dynamics of queries of the concepts of hypoxia and hypoxemia is shown in Fig. 2.
All these search queries, as well as the increased interest of scientists in the study of hypoxia, are currently associated with two factors. The first factor that determines the interest in hypoxia is associated with the discovery of the HIF factor, due to which cells sense and adapt to the presence of oxygen [[1], [2], [3]]. This groundbreaking study was deservedly awarded the 2019 Nobel Prize in Physiology or Medicine (William G. Kaelin, Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza).
The second factor is the global pandemic of coronavirus disease (COVID-19), which has become the greatest modern threat to public health [[4], [5], [6], [7]]. The SARS-CoV-2 virus primarily attacks lung tissues due to impaired alveolar gas exchange in cells, which leads to acute respiratory distress syndrome and systemic hypoxia [[8], [9], [10], [11], [12], [13], [14]]. Hypoxia and hypoxemia are experienced not only by patients with COVID-19, but prolonged self-isolation leads to the development of hypoxia. Therefore, hypoxia is of interest not only to scientists, but also to the general population.
The term “hypoxia” (from the Greek “hypo” – under, below and the Latin “oxygenium” – oxygen) was first proposed by the American physiologist Carl Wiggers in 1941 [15] instead of the previously used term “anoxemia” (from the Greek “an” – negative particle, Latin “oxygenium” – oxygen and Greek “háima” – blood). Note that the first publications in Pubmed date back to 1945. It is known that hypoxia is a condition characterized by a decrease in molecular oxygen (O2) or is associated with a problem of regulation of the oxygen level during intracellular redox reactions [16]. In each of these situations, signs of oxygen starvation of varying severity appear in the body [[17], [18], [19], [20], [21], [22]]. It should be noted that manifestations of oxygen deficiency can also occur due to ischemia of tissues and organs. However, according to the pathogenetic mechanism of its formation, ischemic conditions are manifested not only by a decrease in the O2 index in the affected biological tissues. In many ways, they are also due to problems with the delivery of energy substrates to them, especially glucose [[23], [24], [25], [26], [27]]. Based on this, ischemia should be considered as a more serious and dangerous phenomenon. Against its background, the energy potential of cells decreases much faster. This can be explained by a crisis in ATP production in the mitochondrial compartment [[28], [29], [30], [31], [32]]. For the most part, in studies on the general and local effects of hypoxia on the body, the phenomenon of oxygen deficiency is considered from the point of view of pathology. Hypoxia is usually characterized in scientific articles as a common pathological process [33]. However, during the implementation of different modes of activity, mammals can feel the influence of physiological hypoxia. So, for example, oxygen deficiency occurs in the muscles under the condition of their prolonged contraction [34,35]. The decrease in the content of O_2 in the brain is also associated with mental stress [36,37]. Signs of hypoxia can sometimes be found in organs with a relatively low level of metabolism (liver, kidneys, gastrointestinal tract) [38].
Thus, hypoxia is an interesting topic from the point of view of studying not only pathological, but also adaptive processes. Experimentally modeled hypoxia under controlled conditions in vivo allows understanding the internal mechanisms and testing therapy to accelerate adaptation and alleviate pathological conditions associated with hypoxia. Although in vivo animal models cannot replicate some of the features of human physiology, this makes it possible to predict the behavior of the human body under stress.
There are a certain number of scientific papers in which the authors consider the modeling of hypoxia in animals [[39], [40], [41], [42], [43], [44], [45], [46]]. The authors of [39] pay attention to understanding the topic of cardiometabolic consequences of chronic hypoxia in animal models. Sun et al. provide a review of animal species used in the study of hypoxia-induced seizures and a review of the methodology for seizure induction [44]. A review [45] focuses on the development of a small animal model that incorporates exposure to chronic intermittent hypoxia to create systemic hypertension similar to that seen in humans with obstructive sleep apnea syndrome.
The above works are more focused on modeling some types of hypoxia in animals. The aim of our review was to analyze the information to develop extended and generalized recommendations based on scientific studies of methods for modeling hypoxia in rats, covering methods for modeling endogenous and exogenous hypoxia. The purpose of this work is to systematize the existing methods for modeling hypoxia in rats, their typification, formulation of recommendations for reproduction and warning about limitations in use.
2. Methodology of literature search and selection
The major portion of the literature is devoted to the methodological and technical aspects of modeling hypoxia in rats. In expanding the search, we paid attention to the reproducibility of these methods and the frequency of their occurrence in other scientific literature. As a result of the analysis, we were interested in the adequacy and simplicity of the methods, relevance from the point of view of today. We have made generalizations and groupings according to the physical characteristics of reproduction and the medical purposes of modeling. In the course of the analysis of the literature, in some cases, reservations were formulated regarding the adequacy of the models and the objectivity of the results of the study. The arguments presented in the article will allow researchers to pay attention and avoid errors when reproducing models.
The various databases used for literature searches include databases for original published journal articles and evidence-based databases for integrated information available in the form of systematic reviews and abstracts. Most of them are not free for individual users. But in many, even abstracts allow us to consider the model of hypoxia in Shursa. This made it possible to pay attention to the main parameters of the model and the results achieved. In some cases, we have asked authors to provide hypoxia simulation functions through ResearchGate for a more detailed study of the method. PubMed (https://pubmed.ncbi.nlm.nih.gov), Medline (https://www.nlm.nih.gov), Embase (https://www.embase.com) and Cochrane Central were searched. Register of Controlled Trials (CENTRAL) (https://www.cochranelibrary.com).
2.1. Rodent hypoxia models
Rodent models are the most common models in the simulation of hypoxia. And the rat models dominate over the mouse models. In particular, in “pubmed” for the query “hypoxia + rat” 35,148 results were returned, and for the query “hypoxia + mice” - 26,959 results.
One of the most obvious differences between rats and mice that make them popular in simulations is size and weight: adult rats weigh about eight to ten times as much as mice. This results in a number of practical benefits for research, especially with regard to surgical procedures, ease of processing results, sampling, and volume. From a physiological point of view, the reactions and pathways of development of hypoxia in rats are better known, and a large amount of data has been accumulated over many years. Some results indicate that rats more closely mirror human physiology than other species, and in many cases more closely mimic human disease. The only current advantage of mice in simulating hypoxia is cost. However, significant behavioral differences between the lines of rats, different mobility and adaptation make it necessary to pay attention to the lines when modeling one or another type of hypoxia [47].
It should be noted that the choice of a rat strain for research is important when modeling hypoxia, since different strains of rats demonstrate noticeable differences in reactions to hypoxia [48]. There are several more lines that model hypoxia, such as: Sprague-Dawley, Wistar, Brown Norway, Long Evans, Fisher, Lewis et. al. The lines of Sprague-Dawley and Wistar rats are the most common for modeling hypoxia. At the time of writing, there were 7193 Wistar articles and 10,925 Sprague-Dawley articles in the PubMed library. Other strains of rats are less popular among researchers. The researchers tracked the differences in physiological and adaptive parameters when modeling both hypobaric and normobaric hypoxia. In Table 1 shows the main differences between Sprague-Dawley and Wistar rats when modeling hypobaric hypoxia.
Table 1
Critical ascent height and lifetime at critical height of Sprague-Dawley and Wistar rats in simulation of hypobaric hypoxia.
| Hypobaric characteristics | Sprague-Dowley | Wistar |
|---|---|---|
| Critical altitude lethal altitude | 9250–10668 m altitude [[49], [50], [51], [52], [53]] | 9754–11500 m altitude [[57], [58], [59], [60]] |
| High tolerancea to hypoxia rats (HHT) | More than 20 min [52,[54], [55], [56]] | More than 25 min [57] |
| Medium (normal) tolerancea to hypoxia rats (HHN) | 10–20 min [52,56] | 10–25 min [57] |
| Low tolerancea to hypoxia rats (hypobaric hypoxia Susceptible-HHS) | Less than 10 min [55,56] | Less than 10 min [57] |
It is worth pointing out that Wistar rats were lifted to altitude of 10,000 to 11,500 m [60]. At an altitude of 10,000 m, 38% of animals died, at an altitude of 10,500 m - 83%, and at an altitude of 11,000–91%. Thus, for the correct interpretation of the results, it is necessary to determine in advance the tolerance of rats to hypoxia.
At the same time, it was found that adaptation properties to altitude and biochemical indicators do not change between rats of the Sprague-Dowley and Long Evans lines [61]. But significant differences are described between Sprague-Dowley and Long Evans strains under simulations of containment conditions [62]. In particular, it was established that depending on the strain, oxidative stress and hormonal reactions to chronic periodic hypoxia change. Jin et al. reported that the Sprague Dawley rat strain developed significantly less vascular reactivity than the Wistar rat strain. This points to the role of genetic predisposition to response to hypoxia [63,64]. In addition, in strains of Sprague Dawley and Brown Norway rats, the pressure in the pulmonary arteries during an 8-h constant daily hypoxia for two weeks is different [65]. Brown Norway show a blunted response to hypoxia [66] In endogenous models, the choice of rat strains used for modeling is also important. When modeling respiratory hypoxia (asthma), it should be taken into account that Wistar, Sprague Dawley, Fisher and Lewis strains do not always get an allergic reaction with the production of IgE [67,68]. Despite this fact, Wistar and Sprague Dawley rat models of asthma have been reported [69,70]. Brown Norway, in contrast, is an atopic strain prone to developing IgE reactions after sensitization to an allergen [[71], [72], [73]], therefore this strain is most suitable for studying hypoxia caused by allergic asthma.
It should be noted that the results are also influenced by the social conditions of detention. The above results inform the scientific community that the responses of different strains of rats to simulated hypoxia can vary significantly, which can lead to inaccuracies in predicting results from one study to another. To avoid this, it is necessary to indicate weight, gender, conditions of detention, social conditions of detention (single cages or group keeping), lighting, nutrition, weight, age, health. All these moments can change the result.
Based on the foregoing, the selection and preliminary testing of rat strains for research in this area are important when comparing rat strains with different models of hypoxia.
2.2. Hypoxia. Simulation methods
Hypoxia simulation methods can be divided into two types depending on the types of hypoxia: exogenous and endogenous. The classification of hypoxia modeling methods is shown in Fig. 3.
2.3. Exogenic hypoxia simulation techniques
Exogenous hypoxia occurs when external factors affect the oxygen system. For example, changes in the oxygen content in the inhaled air, changes in the total barometric pressure. Exogenic simulation techiques are divided into hypobaric and normobaric methods.
2.4. Hypobaric hypoxia
Hypobaric hypoxia is formed as a result of a decrease in the partial pressure O_2 in the surrounding air, associated with a change in the barometric pressure of the air, which is accompanied by a decrease in this indicator in the alveolar air. At an altitude of 3000 m above sea level, a reduced oxygen concentration causes high-altitude stress and hypoxia [74,75]. These studies show that residents living above 3000 m may experience damage to the heart, brain, lungs, and other organs due to low levels of inhaled PO_2 and reduced barometric pressure. Therefore, the study of hypobaric hypoxia begins precisely from this height. The body has many mechanisms to regulate oxygen delivery and maintain adequate oxygenation, including respiratory rate, cardiac output, stroke volume, hemoglobin concentration, systemic capillary dilation with pulmonary capillary constriction, and alveolar dilation. A careful balance is maintained through the coordination of all these systems; however, the cellular response plays an important role in how to deal with hypoxia at the cellular level, and this is where disease begins. Exposure to a low oxygen environment at a simulated altitude of 3 km for 8 weeks causes pathological remodeling of the pulmonary vascular walls and pulmonary hypertension, and also leads to a number of pathological changes, including right ventricular hypertrophy [76]. This model is easy to replicate because it is a highly reproducible model and also provides evidence for clinical drug trials. Accordingly, the saturation of hemoglobin with oxygen decreases, which leads to a decrease in the level of O_2 in arterial blood with a simultaneous decrease in the oxygen-venous gradient [77].
Hypobaric hypoxia in rats is reproduced by lowering the barometric pressure of the environment [[78], [79], [80]]. The model reproduces the conditions of a hypobaric state. Stimulates the “rise” of animals in the flow chamber. With the help of this model, they study: the maximum lifetime; threshold at high altitude; adaptive: (proteins of adaptation to hypoxia), namely hypoxia-induced factor-1α (HIF-1α) and myoglobin (Mb); changes in physiological, behavioral, biochemical parameters; immune response to hypobaric hypoxia, the pathogenesis of pulmonary hypertension induced by hypobaric hypoxia is possible.
Industrial pressure chambers are available for simulation, or you can design a homemade pressure chamber for test methods. The pressure chamber walls are made of transparent plastic/glass. This will allow observing the behavior of the animals while performing the protocol. The chamber must be sealed. Relative vacuum (low pressure) in the chamber is created by a rotary vacuum pump by adjusting the inlet air flow rate with an inlet micrometer valve [81]. The internal pressure is monitored by two differential pressure sensors. They are connected to a vacuum regulator driving a diaphragm pressure regulator. Once the desired pressure level has been reached, the chamber's internal barometric pressure must be adjusted and maintained. The oxygen level inside the pressure chamber is monitored by gas analyzers. The chamber is periodically purged with air to maintain a normal gas ratio and avoid hypercapnia. CO2 levels [82] can be controlled with soda lime. At the bottom of the chamber was placed 50 g of granulated soda lime; Two weeks before the experiment, rats should be selected to determine their individual sensitivity to the state of hypoxia [83, 84]. As already mentioned, the height of this area is different in different strains of rats. From this point on, atmospheric pressure should recover to its original level. According to the time spent at altitude, animals are divided into high- and low-resistant to hypoxia. Such a distribution will prevent a strong scatter in the experimental sample. For experiments, it is better to take low-resistant animals, since the effect of pressure in these animals will be much more pronounced. In this case, the rate of “rise” directly depends on the rate of pumping air out of the chamber. Depending on the goals of modeling, the rate of “rise” should be different.
2.5. Normobaric hypoxia
Normobaric hypoxia is associated with a decrease in the percentage of oxygen in the inhaled air due to a change in its gas composition at a normal barometric pressure of 750 mm Hg (105 Pa); insulating hypercapnic hypoxia is retated to both: a decrease in the oxygen content in the recirculating air mixture and its absorption during breathing, and an increase in the partial pressure of CO2 due to the accumulation of exhaled air [85, 86]. By means of simple calculations, we note that the hypoxic oxygen concentration in such a mixture should not be greater than ∼14.4%. It is this concentration of oxygen in the mixture that corresponds to 3000 m (14.4% of oxygen in the mixture exerts the same partial pressure as oxygen at an altitude of 3000 m above sea level). After all, it is precisely at 3000 m, and accordingly at an oxygen content of 14.4%, that signs of hypoxia begin to develop in unadapted animals.
Hypoxic normobaric hypoxia is based on the use of hypoxic gas mixtures and membrane hypoxicators. They are used to study both acute and chronic hypoxic exposure, hypoxic training, evaluation of rehabilitation processes, etc. Normobaric methods have 2 branches: dynamic and static. For these techniques, it is also necessary to have a sealed chamber with transparent walls that allow observation of the animal. Let us give examples of dynamic and static normobaric methods.
The division into static and dynamic hypoxia is exclusively related to different physical methods of achieving hypoxia. But the hypoxia that can be achieved by these methods from a biological point of view can be divided into acute, intermittent and chronic with or without hypercapnia.
2.5.1. Dynamic normobaric techniques
(a) Hypoxic hypercapnic hypoxia. It is achieved by creating a variable microspace in the containment system by saturating the exhaled air [[87], [88], [89], [90]]. There is no external adjustment of the oxygen concentration. Only oxygen depletion of the habitat is observed. Technically, this model is very easy to reproduce, but its complexity lies in differentiating hypoxia from hypercapnia. Such techniques make it possible to observe the adaptation of animals to hypercapnia. The main goals of observation are acute hypoxia with hypercapnia [91,92]. They are also used to confirm the antihypoxic activity of drugs. To better understand what the experiment looks like, let's describe one of them [93] periodic hypercapnic hypoxia is achieved in an isolated chamber by periodically changing the levels of O2 and O2 at a rate of 5 cycles per hour for 24 h. The parameters for each 12-min cycle were 2.4 min at 10% O2/6% CO2 and 9.6 min at 21% O2/0% CO2 . By adding to that experiment in the cycle an interval with increased oxygen in the absence of CO2, it is possible to achieve the effect of hyperoxia on hypoxia and hypercapnia. Then the proposed cycle time will be as follows: 2.4 min at 10% /6% CO2 , 4.5 min at 30% O2/0% CO2 and 5.1 min at 21% /0% .
(b) Create an environment with a specific CO2 concentration and varying O2 concentration in the flow chamber by creating cycles. These techniques include techniques for achieving acute/periodic hypoxia. Acute/intermittent hypoxia is created by reduced O2 concentration balanced by . Hypoxia with normobaria is created with a certain frequency. For example, chronic intermittent exposure to hypoxia was investigated [94]. The intermittent hypoxia exposure profile consisted of 180s periods, which in turn contained 90s intervals of 10% , which alternated with 90s intervals of 21% .
- ❖By alternating concentrations and different cycles and modes, we can achieve various hypoxic effects, which can be conditionally divided as:
- ❖Episodic hypoxia is a regime where prolonged normoxia is interrupted by short episodes of hypoxia.
- ❖Intermittent hypoxia - prolonged hypoxia is interrupted by short episodes of normoxia.
- ❖Adaptive hypoxia can be achieved gradually, i.e. the concentration of in the mixture gradually decreases with normoxia, the concentration of does not decrease.
2.5.2. Static normobaric technique
In static normobaric procedures, animals are kept at a constant oxygen concentration for a long time. The rats are placed in the chamber. Soda lime is placed at the bottom of the chamber. and cylinders are connected to the chamber, creating a microclimate in the chamber with a constant low oxygen concentration. Animals were placed in a custom-made 16-L plastic apparatus to create normobaric hypoxia [95]. Fresh soda lime was placed at the bottom of the chamber. and cylinders were connected to the chamber. The concentration was monitored by infusion at a flow rate of 7.5 L/min. The concentrations of and were monitored continuously, respectively, concentrations of 18%, 15%, 12%, 10%, 8%, 6% were used for . Physiological responses to hypoxia, both continuous and intermittent, depend on the -sensing capacity of the chemoreceptors of peripheral arteries, especially the carotid bodies, and the following reflexes play an important role in maintaining homeostasis. With the help of dynamic (periodic) hypoxia, sleep apnea is studied in rat models. Although we included sleep apnea in the classification of respiratory hypoxia (because there are other methods of modeling this disease), the periodic hypoxia method remains the most popular. In particular, sleep apnea was achieved by periodically cycling oxygen levels between 10 and 21% every 90 s, i.e. 40 cycles/h [96]. This model has demonstrated its effectiveness in reducing pO 2 values during hypoxic cycles [97].
All exogenous methods are technically difficult, as they require appropriate equipment. Still, with their help, one can study the complex effect of hypoxia on the entire body, and understand the redistribution of oxygen between tissues during pathology. These methods make it possible to determine the effect of hypoxia on the brain and cardiovascular system without introducing specific drugs that can also have an effect. They do not require additional use of medical drugs, which excludes moments of individual reactions. These are comprehensive and well-reproducible methods that provide good informativeness. Exogenous methods cannot reproduce upper airway occlusion, intrathoracic pressure fluctuations, excitation, and controlled hypercapnia. On the other hand, reproducing a single clinical characteristic by exposure to exogenous hypoxia appears to be relevant to induce pathophysiological changes similar to the clinical manifestations of patients suffering from hypoxia, such as endothelial dysfunction, atherosclerosis, arterial hypertension, pulmonary hypertension, and heart failure [98,99].
2.6. Endogenic hypoxia simulation techniques
2.6.1. Modeling of respiratory hypoxia
Hypoxia in these cases is considered as a criterion for achieving pathological conditions, rather than as an independent factor. However, it should be noted that there is no ideal model that would fully mimic human pathology and would include all these features of pulmonary respiratory pathology [19]. In experimental studies, it is best to choose those models in which hypoxia is best manifested. The main models of respiratory hypoxia are the model of bronchial asthma, acute pulmonary edema, apnea, and acute lung injury. Animal models, in particular rats, should mimic one or more physiological and pathological mechanisms and results of respiratory pathologies in humans (detection of rapid development measured in hours, impaired gas exchange, decreased lung compliance, increased permeability of the alveolar-capillary membrane).
2.6.2. Simulation of bronchial asthma
In rats, asthma does not develop spontaneously. The disease occurs artificially in the respiratory tract. Allergens are used in experimental models of deseases, including ovalbumin (OVA). In addition to OVA, there are other allergens used in animal models: house dust mites (dust mites) (HDM), such as Dermatophagoides pteronyssinus (Der p) or D. farinae (Der f), as well as mite allergens (Der p 1, Der f 1, Der p 23, etc.), fungi (Aspergillus fumigatus, Alternaria alternate), cockroach extracts, roundworm antigens, cotton dust, ragweed and latex (Hevea brasiliensis). The choice of allergen depends on the condition to be reproduced and can be used alone or in combination, as they all cause different immunological and non-immunological mechanisms involved in the pathogenesis of asthma [[100], [101], [102], [103]].
OVA is the most widely used allergen. It is obtained from chicken eggs, which allows it to be produced in large quantities, which reduces the cost of the experiment. OVA is used in experimental models of asthma and causes intense allergic pneumonia. The only caveat to using OVA is that OVA does not cause airway inflammation in humans, and therefore its use to study asthma is sometimes questioned. Because humans and rats diverged as separate species millions of years ago, they have adapted to allergens specific to their unique ecological niches, so it is not surprising that there are differences in the innate immune response of humans and rats to allergens. The OVA models developed differ from the OVA sensitization protocol in terms of route of administration, adjuvants, level of sensitization, and the number of calls made. For example, rats are administered OVA subcutaneously [104], and in other literature intravenously [105]. OVA is adsorbed on Al(OH)3 gel, which acts as an adjuvant at different concentrations of 1:200 [104] and 1:100, depending on the desired effects [105]. Introductions are made on the 1st day, 7th day, 21st day [106], or only on the 1st day [104]. To enhance the effect at some stage begin to carry out inhalation of OVA at a volume weight concentration of 1–5% w/v with a certain interval and repetitions.
It was also found that concomitant administration of lipopolysaccharide induces the phenotypes of severe asthma with eosinophilic, neutrophilic, and lymphocytic inflammation [107]. A neutrophilic inflammatory response with an increase in intrapulmonary cytokines is manifested in the introduction of LPS [108,109]. Changes in alveolar-capillary permeability are moderate. Good reproducibility of asthma with parallel administration of OVA and LPS is noted. After a certain number of days after the start of the experiment, the animals are removed from the experiment with venous blood collection, removal and fixation of tissue biochemical parameters, and the percentage of blood oxygenation.
Rat models remain the easiest way to understand the pathophysiology of allergic asthma and to help develop new drugs and immunotherapy strategies to treat this complex disease. Among the major objections to rat models of asthma is that they do not mimic real-world ways of inducing an allergic response. Asthma is a chronic disease that occurs as a result of periodic or long-term exposure to aeroallergens, which leads to inflammation of the respiratory tract. This exposure occurs throughout life, mainly through the inhalation of allergens and irritants through the respiratory tract during ventilation. In addition, the use of intraperitoneal or subcutaneous routes to sensitize animals is far from natural in terms of inducing an allergic inflammatory response. Therefore, to improve these models, it is necessary to develop protocols that use natural routes of administration for humans (in particular, the airways), and not use the allergens themselves that cause clinical disease (eg, HDM).
2.6.3. Simulation of acute pulmonary edema
Pulmonary edema is defined as a condition with excess extravascular pulmonary fluid. It usually occurs due to increased hydrostatic pressure or hyperpermeability in the pulmonary capillary bed [110,111]. Simulation of acute pulmonary edema can be achieved by both pharmacological and surgical methods. Consider the most relevant of them for today.
The main model of acute pulmonary edema is the angiotensin model. This model received additional attention due to damage due to COVID-19 infection. In particular, SARS-CoV-2 and SARS-CoV-1 viruses disrupt the structure of the alveoli, which is the leading cause of pulmonary pathology, characterized by increased fluid permeability, cell death, and inflammation, as well as decreased gas exchange and surfactant levels. A plausible hypothesis of SARS-CoV-2 damage to the lung parenchyma is that the virus infects pneumocytes by binding to the angiotensin-converting enzyme (ACE-2) receptor, leading to decreased angiotensin II conversion and reduced angiotensin [112] and its effects. Accordingly, angiotensin II levels increase in the alveolar microenvironment, which can potentially affect different types of cells. Edematogenous dose of AT-II is administered intravenously to rats. A 10 μg/ml solution of angiotensin II (AT-II) in saline pH 6–7 at 1 ml/kg is a sufficient edematogenous dose to induce pulmonary edema in rats [113].
The next most commonly used model is the adrenaline model. Edematogenous adrenaline dose for rats ranges from 25, 50, or 100 mcg/kg. Adrenaline induces plasma kallikrein activation, kininogen consumption, and kin in release in rat blood in vivo and in vitro [114]. These changes are accompanied by pulmonary edema. The vasoactive substance (adrenaline) leads to the development of pulmonary edema, which is manifested by a characteristic clinical and morphological picture, a sharp decrease in blood oxygenation, as well as changes in weight coefficient (WC) and dry matter (DM). An increase in WC and a decrease in DM indicate the presence and severity of pulmonary edema.
Other substances to achieve experimental pulmonary edema may be intravenous injections of 0.1 ml/kg of high-frequency oleic acid in dissolved saline (0.1 ml/kg) [115]. Oleic acid is directly toxic to endothelial cells at concentrations of 5 × 10–4 M in vitro [116]. Endothelial damage is accompanied by epithelial damage with edema and necrosis of type I cells [117]. Therefore, this method can also be included in the methods of acute lung injuries. Pulmonary edema is also caused by injections of 14 mg/kg of lipopolysaccharides (LPS) into the caudal vein [118].
LPS is a glycolipid present in the outer membrane of gram-negative bacteria, which consists of a polar lipid head group (lipid A) and a chain of repeating disaccharides [119]. Most of the biological effects of LPS are reproduced by lipid A [120], although the presence or absence of repetitive oligosaccharide O antigen affects the magnitude of the response [121]. LPS is an important mediator of sepsis in response to gram-negative bacteria, and systemic administration of LPS was one of the first approaches used to model the effects of bacterial sepsis. Therefore, it is also used in the modeling of lung damage. LPS produces a neutrophilic inflammatory response with increasing intrapulmonary cytokines while changes in alveolar-capillary permeability are moderate. In addition, the lungs of rats can cause severe pulmonary embolism [122]. Examples of surgical models of acute pulmonary edema include lung transplantation, described in detail in Ref. [123].
Models of circulatory-respiratory ischemia of the lungs are mainly focused on the lesions of the epithelium and endothelium of the inner surface of the alveoli. Ischemia with subsequent reperfusion (I/R), either in the lungs or in distant vascular channels, can lead to lung damage. This is a combined model. Lung transplantation is a classic form of injury resulting from lung I/R. This reaction is not associated with pulmonary rejection [124,125], characterized by noncardiogenic edema, inflammatory infiltrates, and hypoxia. It is not necessary to transplant the lungs for modeling in rats. The lungs are equipped with two separate vascular systems, pulmonary circulation, and bronchial circulation. Ischemia can be caused by squeezing the pulmonary artery, which maintains bronchial circulation, or squeezing the trachea, which stops all blood circulation. In the variant of the I/R model of the lung, ischemia is achieved by the collapse of the lung with subsequent re-expansion [126].
The use of bacterial LPS has a number of advantages as a method of modeling the effects of gram-negative bacteria on humans and animals. But while humans have average sensitivity to LPS, rats have low sensitivity [127]. LPS is easy to apply and the results are generally reproducible in experiments. LPS is a potent activator of innate immune responses through TLR4 pathways and has little direct toxicity to cells in vitro. Thus, the use of LPS provides information on the effects of host inflammatory responses that occur during bacterial infections. However, LPS has some significant drawbacks. LPS preparations vary in purity and may be contaminated with bacterial lipoproteins and other bacterial materials [128]. In general, simulated LPS do not cause the severe endothelial and epithelial damage that occurs in people with SARS [129]. Thus, LPS alone gives an incomplete picture of the effect of live bacteria on the lungs.
A major advantage of nonpulmonary ischemia-reperfusion models is that they reproduce a known clinical phenomenon, the development of lung injury after intestinal or peripheral ischemia and reperfusion in humans. However, when the I/R of non-pulmonary vascular beds is the only influencing factor, lung injury is mild and secondary stimuli (such as LPS) are required to induce significant lung injury. In addition, abdominal surgery is necessary for the intestinal I/R model, which induces an inflammatory response that needs to be controlled experimentally.
2.6.4. Simulation of apnea
Chronic intermittent hypoxia is one of the hallmarks of sleep apnea. For one of the methods of its simulation is the overlap of the tracheotomy cannula, which is closed for at least 10 s (simulation of apnea). The rat model of the upper airway occlusion (тracheostomy implantation) is performed under anesthesia [130]. Tracheotomized and anesthetized rats were ventilated via a computer-controlled collapsible upper airway segment [131]. Occasional hypoxia observed in sleep apnea contributes to the daily increase in blood pressure. Crossland et al. achieved apnea by remote inflation of a balloon or an obstruction device implanted in the trachea in freely moving rats [132]. Liu et al. to simulate obstructive sleep apnea, Sprague-Dawley rats were injected with 0.1 ml of sodium hyaluronate solution in the upper respiratory tract at the junction between the hard and soft palate [133]. This model resulted in morphological and physiological changes in the heart and lungs due to hypoxia. It should be noted that these models are labor-intensive and have low throughput.
2.6.5. Model of acute lung injury
2.6.5.1. Acid aspiration
The acid component of gastric aspirates is often modeled by intratracheal instillation of hydrochloric acid (HCl) in animals. Lung damage in rats after GA of dilute hydrochloric acid (ACID = normal saline (NS)+ HCl, final pH 1.25) [134] occurs in a two-phase reaction, including an early lesion characterized by stimulation of capsaicin-sensitive neurons and direct caustic action low pH on the airway epithelium followed by an acute neutrophilic inflammatory response after 4–6 h [104]. These pathogenic mechanisms lead to the loss of microvascular integrity of the lungs and the extravasation of fluid and protein into the airways and alveoli [135,136]. For modeling rats are anesthetized with 5% isoflurane in oxygen at a rate of 5 l/min. After induction of anesthesia, rats were injected with NS + HCl, pH 1.25, by deep oral injection into the trachea.
The acid aspiration model is particularly useful for studying hemodynamic and physiological changes in acute lung injury, ventilation strategies, and mechanisms of neutrophil recruitment. Acid aspiration can also be used in combination with other techniques, such as mechanical ventilation, to further replicate clinically relevant scenarios [137]. A limitation of the acid aspiration method is that the difference between harmful and harmless acid concentrations is very small. Another limitation is that humans do not aspirate HCl, but instead aspirate complex gastric contents, which are a suspension of solids, bacterial products, cytokines, and a pH that is typically greater than 1.5. Further studies are needed to determine whether these differences between gastric fluid and HCl result in a difference between this model and the damage seen in humans after the aspiration of gastric contents.
2.7. Mechanical ventilation
During the 1980s and 1990s, several studies showed that artificial lung ventilation could cause pneumonia and injuries in animals called ventilator-induced lung damage [138,139]. Detachment of endothelial cells from the basement membrane and death of epithelial cells with exposure to the epithelial basement membrane becomes apparent after 20 min of artificial lung ventilation with very large tidal volumes (formation of a peak pressure of 45 cmH2O in rats or 4,41 kPa) [140].
The main advantage of the mechanical ventilation model is its clinical relevance, which is emphasized by the fact that it is the only model that has led to changes in clinical practice. The main drawback is the complexity of the model. Furthermore, animals such as rats can only be ventilated for a relatively short period of time, whereas patients require mechanical ventilation for days or weeks.
2.8. Blood (hemic) hypoxia
Acute hemic hypoxia is based on a decrease in blood oxygen capacity. It is caused either by a change in the properties of hemoglobin (for example, the conversion of hemoglobin into carboxyhemoglobin or methemoglobin) or by a decrease in the amount of hemoglobin (bloodletting).
2.8.1. Simulation of anemic hypoxia
Anemia in rats can be obtained by replacing the blood with an equal amount of lactated Ringer's solution through the caudal artery [141]. Similarly, for the simulation of anemic hypoxia, isovalemic hemodilution is performed with rat plasma. Blood is drawn from the femoral artery, while plasma is injected into the left jugular vein at a rate of 8 ml/h using an infusion pump. Standard 40 μm filters are used to prevent the transfusion of aggregates or cellular debris [142].
2.8.2. Carboxyhemoglobinemia
Carbon monoxide () is a colorless, odorless, and tasteless gas that is toxic when it binds to hemoglobin to form carboxyhemoglobin, blocking the delivery of oxygen () to tissues. To study carboxyhemoglobinemia animals are kept in an airtight chamber. Rats aged one to ten weeks were exposed to carbon monoxide at concentrations of 150, 250, 500, and 2400 ppm for up to 240 min [[143], [144], [145]]. Spectrophotometry is used to determine the carboxyhemoglobin content.
2.8.3. Methemoglobinemia
Methemoglobin is a form of oxidized hemoglobin that is unable to react with oxygen and transfer it to all organs of the body. Such compounds that cause methemoglobinemia include, for example, . A single dose of intraperitoneally in different dosing conditions from 25 to 75 mg/kg body weight (dosing volume 2 ml). Subcutaneous injection of solution is allowed [146]. With the intra-abdominal route of administration, 100% of death of the animal occurs after 13–17 min, with the subcutaneous route of administration - after 27–30 min. If you need to simulate mild (methemoglobin blood 18–20%) hemic hypoxia, rats are injected subcutaneously with sodium nitrite () at a dose of 3 mg/100 g body weight, to simulate moderate hypoxia (methemoglobin 35–36%) is used intraperitoneally sodium nitrite at a dose of 5 mg/100 g body weight. The simulation was used to diagnose and treat methemoglobinemia.
Models, developed for simulating the kinetics of carbon monoxide and sodium nitrite in humans are adapted for use in animal models. They provide significant information for clinicians on the development of hemic hypoxia in humans.
2.9. Simulation of cardiovascular (circulatory) hypoxia
Hypoxia in this case is considered solely as a complication of pathological conditions, not as an independent factor. Its main symptom is impaired blood supply to tissues. Therefore, circulatory hypoxia can be locally concentrated and common to the whole organism. All types of ischemia can be attributed to local circulatory hypoxia. Pulmonary ischemia will be caused by hypoxia of the whole body (described above). Conditions that affect the whole body leading to hypoxia include cardiogenic ischemic conditions. In particular, circulatory disorders violate the pumping function of the heart (acute and chronic heart failure in myocardial infarction, hemodynamically significant acute cardiac arrhythmias).
2.9.1. Simulation of chronic heart failure
For modeling chronic heart failure as well as for respiratory hypoxia 2 groups of methods are used - pharmacological and surgical. The period of formation of significant chronic heart failure and circulatory hypoxia in rats is 6–8 weeks. Regardless of the modeling methods, the assessment of the functional state of the heart muscle is carried out by morphofunctional methods, the most informative of which is dynamic transthoracic echocardiography.
2.10. Pharmacological models
Pharmacological methods are aimed at the formation of profound metabolic changes in the myocardium of animals, leading to a steady decrease in their contractile capacity. The most common are cardiotoxic drugs such as adriamycin (an anticancer drug), isoprenaline (medicines used for bradycardia and heart block), and monocrotaline, a pyrrolizidine alkaloid used to model pulmonary hypertension. According to the literature [147,148] to achieve the effect and development of cardiomyopathy sufficient administration of 1–2 mg/kg of adriamycin to rats per week for 10–14 weeks. From 3 to 23 will show histological changes in the myocardium, and the effects of hypoxia. The most effective dose of isoprenaline, which causes significant myocardial damage together with an acceptable level of survival in rats is 150 mg/kg/day subcutaneously with an interval of 24 h for 2 days [149]. Monocrotalin (MCT) causes pulmonary vascular syndrome in rats, characterized by proliferative pulmonary vasculitis and pulmonary hypertension. This model is based on the metabolic activation of subcutaneous MCT as a pyrrolizidine alkaloid by hepatic cytochrome P450 3A [150,151]. Active pyrrole MCT is pneumotoxic [152]. Pneumotoxic doses are divided depending on the timing of the effects of the study. Doses of 60 or 80 mg/kg are suitable for short-term effects. They cause the death of animals within 3–6 weeks. The study of long-term effects requires the introduction of a lower dose [153]. 40 mg/kg MCT causes acute muscle tissue of the smallest pulmonary arterioles together with high PVR and right ventricular hypertrophy 4 weeks after MCT administration. However, at 8 and 12 weeks after MCT, pulmonary arteriole abnormalities were restored, accompanied by normalization of right ventricular function, although cardiomyocyte hypertrophy persisted [154].
2.11. Surgical models
The basis of the surgical method modeling chronic heart failure is the formation of postinfarction scar and postinfarction cardiosclerosis. The procedure involves opening the pericardium and applying a superficial suture to the epicardium of the left ventricle. The animals underwent permanent (8-week) occlusion of the left coronary artery 2 mm distal to the beginning of the aorta using a modified technique, which leads to a large infarction of the left ventricle. Eight weeks after surgery, parameters indicating chronic heart failure are measured [155].
2.11.1. Simulation of acute histotoxic hypoxia
Acute histotoxic hypoxia (also called histoxic hypoxia) is based on the direct interaction of various poisons with cytochrome oxidase - an enzyme of the terminal part of the respiratory chain, which leads to the suppression of its activity [156]. Typical cytochrome oxidase inhibitors are hydrocyanic acid (), its salts (, ), and sodium nitroprusside, which lower blood pressure (). Acute histotoxic hypoxia is simulated by intraperitoneally administering 0.4% aqueous sodium nitroprusside solution (3.8 ± 0.1 mg/kg) to rats [157]. Then record arterial blood counts and heart rate of oxygen pressure in tissues.
Infusion of sodium nitroprusside increases cardiac output and total oxygen transport in rats, it worsens the mismatch of ventilation and perfusion. Sodium nitroprusside (SNP) is widely used as a potent vasodilator and donor of nitric oxide (NO), while the cytotoxicity of SNP is well documented. Because the sodium pump () plays a role in regulating smooth muscle contractility, changes in enzyme activity due to hypoxia may contribute to the mechanism of hypoxic narrowing of pulmonary vasoconstriction [158,159].
2.11.2. Simulation of prenatal hypoxia
Side effects of fetal hypoxia include restriction of intrauterine development and prenatal morbidity and, as a result, mortality. The study was performed on pregnant female rats exposed to hypoxia (10.5% ) by continuous infusion of nitrogen gas and a mixture of compressed air. is removed by circulating the atmosphere through sodium lime, and the water contained in the exhalation is retained in a cooled glass tank. Females are subjected to hypoxia for 6 days during 15–21 days of pregnancy [160,161].
2.12. Tissue hypoxia
2.12.1. Simulation of local hypoxia
Simulation of local hypoxia of vital organs is used to confirm the effectiveness and specificity of the action of antihypoxants on the functions of various organs in the simulation conditions close to clinical pathology. Hypoxia is modeled by the ligation of vessels or the temporary imposition of clamps on them. Ischemia is modeled in the same way by the introduction of foreign bodies (emboli) into the lumen of blood vessels, and the formation of emboli in vivo. Temporary clamping or ligation of the artery, intravenous pituitrin, or joint administration of pituitrin and isadrine.
2.12.2. Simulation of cerebral ischemia
Animal models of focal cerebral ischemia are used to study the pathogenesis and possible treatment strategies for human stroke. Because ischemic stroke is often caused by occlusion of the middle cerebral artery or one of its branches, a model of its isolated occlusion - MCAO (middle cerebral artery occlusion) was chosen for the study. MCAO with endovascular filament is a widely used model for inducing focal cerebral ischemia. MCAO is the most commonly used minimally invasive model of stroke in rodents, which allows for controlling the duration and intensity of blood supply for permanent or transient focal ischemia [[162], [163], [164]]. These models are characterized by good reproducibility of infarct size and functional deficit, low mortality, visual confirmation of successful MCAO, and the ability to adapt the model to create infarcts of different sizes and locations. Another popular model is carotid artery occlusion, which was proposed in 1981. However, these models do not allow the study of thrombolytic therapies or studies of drugs designed to target the reperfusion phase after ischemia [165] because they induce permanent focal ischemia. In addition, the induction of MCAO is technically difficult. The rat embolic cerebral ischemia model provides a reproducible and predictable volume of myocardial infarction in the MCA. The MCA was occluded by an embolus in Wistar rats [166]. Formed suspensions of autologous microembolus, resembling arterial thrombi. With the help of the method of continuous flow through the catheter of the carotid artery, it is possible to avoid the reflux of blood with uncontrolled coagulation and embolization, thus ensuring control animals without ischemic lesions [167]. Thus, these models are a powerful tool for translational stroke research. Models of in vivo clot formation is also interesting. The common carotid artery of the rat was irradiated for 6,5 min with an argon laser at 514.5 nm after intravenous injection of a photosensitizer. Retinal embolism was observed in 1 rat 5 min after irradiation [168].
The models shown basically do not reproduce the background pathophysiology that would lead to cerebral ischemia. Thus, we must accept the limitations inherent in these models and follow up early efficacy studies in young, healthy animals with more rigorous studies in age and comorbidity models.
2.12.3. Simulation of myocardial ischemia
The main methods of reproduction in animal experiments of ischemia/myocardial infarction are the following:
❖induction of coronary stenosis [169]. Used in this work apolipoprotein E gene knockout (ApoE KO) rats to induce hypercholesterolemia. This model is well established in the study of atherosclerosis, as rats with apolipoprotein E deficiency (ApoE−/−) show poor clearance of lipoproteins, which in turn leads to the accumulation of particles enriched with cholesterol esters in the blood, which contributes to the development of atherosclerosis. Coronary stenosis is achieved in this case with a special diet high in cholesterol/bile (Pagen's diet)
- ❖stress-induced myocardial ischemia through psychological stress in rats. For example, this is achieved by limiting activity for 6 h, followed by stimulation by clamping the tail for 5 min daily for 14 days [170].
- ❖coronary microembolization [171] is the introduction of a suspension of saline microspheres in the left ventricle during 10s occlusion of the ascending aorta.
- ❖reproduction of ischemic cardiomyopathy by permanent ligation of the left coronary artery in rats [172].
- ❖destruction (ablation) of the atrioventricular conduction system of the heart, such as tamoxifen [173].
- ❖occlusion of coronary vessels is performed by thoracotomy on the left through the fourth intercostal space [174].
- ❖coronary artery ligation [175].
- ❖induction of infarction by administration of substances derived from pyrocatechol, in particular, epinephrine (adrenaline). A subcutaneous single dose of 2 mg/kg of adrenaline is sufficient for this for 2 days [176].
2.13. Hypoxia overload
During physical activity there is a decrease in oxygen, which leads to hypoxia. Rats are subjected to anaerobic exercise. To do this, use a treadmill for rats at a speed of 35 m/min−1, 20 min [177], or swimming [178]. After the experiments, the correlation of HIF-1α and VEGF in brain tissues after anaerobic exercise was tested in rats.
3. Conclusions
Animal models have played a key role in understanding the pathogenesis of hypoxia, its consequences, and treatment. Recent research in this area includes the search for and improvement of models of different types in rats to simulate hypoxia, which will allow the results of these studies to be used and extrapolated to humans. Increasing evidence linking hypoxia to various diseases requires further research to determine the true impact of this disorder. In the future, we advocate increasing attention to clinical realism in hypoxia simulations. Laboratories should attempt to standardize protocols using clinically realistic conditions because exogenous methods, for example, do not actually investigate the effect of temperature and humidity on hypoxia in rats when exogenous methods are simulated. After all, it is known that a high concentration of water vapor in the air also leads to hypoxia. There is a growing recognition that all types of hypoxia are complex and complex pathologies caused by many factors. There is a need to use new methods for monitoring the state of hypoxia in rats, regardless of whether local hypoxia or hypoxia of the whole organism is being investigated. Rodents are uniquely suited to this type of work as invasive, non-invasive, or genetic interventions can be used to explore each of these variables. Without a doubt, the history of research on hypoxia in rat model animals has been fruitful and has brought significant results, but this area is ready for even greater discoveries, especially against the backdrop of new global challenges to humanity.
4. Declaration
Author contribution statement
All authors listed have significantly contributed to the development and the writing of this article.
Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data availability statement
The data that has been used is confidential.
Declaration of interest's statement
The authors declare no competing interests.