Hippocampal alterations after SARS-CoV-2 infection: A systematic review.

SARS-CoV-2 infection produces a wide range of symptoms. Some of the structural changes caused by the virus in the nervous system are found in the medial temporal lobe, and several neuropsychological sequelae of COVID-19 are related to the function of the hippocampus. The main objective of the systematic review is to update and further analyze the existing evidence of hippocampal and related cortices' structural and functional alterations due to SARS-CoV-2 infection. Both clinical and preclinical studies that used different methodologies to explore the effects of this disease at different stages and grades of severity were considered, besides exploring related cognitive and emotional symptomatology. A total of 24 studies were identified by searching in SCOPUS, Web Of Science (WOS), PubMed, and PsycInfo databases up to October 3rd, 2022. Thirteen studies were performed in clinical human samples, 9 included preclinical animal models, 3 were performed post-mortem, and 1 included both post-mortem and preclinical samples. Alterations in the hippocampus were detected in the acute stage and after several months of infection. Clinical studies revealed alterations in hippocampal connectivity and metabolism. Memory alterations correlated with altered metabolic profiles or changes in grey matter volumes. Hippocampal human postmortem and animal studies observed alterations in neurogenesis, dendrites, and immune response, besides high apoptosis and neuroinflammation. Preclinical studies reported the viral load in the hippocampus. Olfactory dysfunction was associated with alterations in brain functionality. Several clinical studies revealed cognitive complaints, neuropsychological alterations, and depressive and anxious symptomatology.

Female students' personality and stress response to an academic examination.

BACKGROUND: Women are vulnerable to stress-related disorders. Examinations are a source of stress, triggering emotional, cognitive, and hormonal responses. We examined women's psychological and hormonal stress responses and academic performance according to personality during a real-life examination. METHODS: Female students (N = 66) were divided into two groups based on hierarchical cluster analysis: one cluster characterized by high neuroticism and moderate extraversion (HN-ME; n = 42) and the other by low neuroticism and high extraversion (LN-HE; n = 24). Academic performance, perceived stress, and emotional dysregulation were analyzed. State anxiety, affect, and cortisol release were measured before and on the examination day. RESULTS: The HN-ME cluster was high in perceived stress, emotional dysregulation, and negative affect. This cluster also had higher state anxiety levels two days before and shortly after the examination compared to the LN-HE cluster. Students' cortisol levels were higher on the examination day, and there was a marginal significance of the Cluster factor in the cortisol release regardless of the day of measurement. CONCLUSIONS: Women with high neuroticism and moderate extraversion may be more vulnerable to psychological stress in academic settings but similar to other women in their cortisol response.

Association between olfactory dysfunction and mood disturbances with objective and subjective cognitive deficits in long-COVID.

Background and purpose: The coronavirus disease 2019 (COVID-19) has been associated with olfactory dysfunction. The persistent symptoms of anosmia or hyposmia were associated in previous studies with the development of memory impairment and mood disturbances. We aimed to investigate the association between the chronicity of reported olfactory dysfunction and subjective and objective cognitive performance in long-COVID patients and to explore whether their emotional symptoms are related to their cognition. Methods: One hundred twenty-eight long-COVID participants were recruited. Reported symptomatology, subjective memory complaints, anxiety and depression symptomatology, and trait-anxiety were assessed. Subjective memory complaints and mood disturbances were compared among groups of participants with olfactory dysfunction as an acute (AOD), persistent (POD), or nonexistent (NOD) symptom. Seventy-six of the volunteers also participated in a face-to-face session to assess their objective performance on tests of general cognitive function and verbal declarative memory. Objective cognitive performance and mood disturbances were compared among the AOD, POD, and NOD groups. Results: The subjective memory complaints and the anxiety and depression symptoms were similar among the groups, but the score in general cognitive function was lower in the participants with symptoms of acute olfactory dysfunction than in those with no olfactory symptoms at any time. Participants' memory complaints were positively related to their emotional symptoms. The relationship between depressive symptomatology and memory complaints interacted with the olfactory dysfunction, as it only occurred in the participants without symptoms of olfactory dysfunction. Depressive symptomatology and acute olfactory symptoms were negatively associated with general cognitive function and delayed memory performance. The months elapsed from diagnosis to assessment also predicted delayed memory performance. Anxious symptomatology was negatively associated with the immediate ability to recall verbal information in participants who did not present olfactory dysfunction in the acute phase of the infection. Conclusion: Olfactory dysfunction in the acute phase of the infection by COVID-19 is related to cognitive deficits in objective tests, and mood disturbances are associated with self-reported and objective memory. These findings may contribute to further understanding the neuropsychological and emotional aspects of long-COVID.

Anosmia in COVID-19 could be associated with long-term deficits in the consolidation of procedural and verbal declarative memories.

Background and purpose: Long-COVID describes the long-term effects of the coronavirus disease 2019 (COVID-19). In long-COVID patients, neuropsychological alterations are frequently reported symptoms. Research points to medial temporal lobe dysfunction and its association with anosmia in long-COVID patients. This study aims to investigate the acquisition and consolidation of declarative and procedural memory in long-COVID patients and to explore whether anosmia is related to these dissociated memory functions. Methods: Forty-two long-COVID participants and 30 controls (C) were recruited. The sample of long-COVID patients was divided into two groups based on the presence or absence of anosmia, group A and group NA, respectively. Objective performance in verbal declarative memory (Paired-Associate Learning, PAL), procedural memory (Mirror Tracing Test, MTT), general cognitive function (Montreal Cognitive Assessment scale), psychomotor speed, and incidental learning (Digit Symbol Substitution Test) were assessed and compared among the A, NA, and C groups. Long-term retention of PAL and MTT were assessed 24 h after acquisition. Results: Lower scores in general cognition, psychomotor speed, and sustained attention were found in A and NA compared with C. However, incidental learning, both cue-guided and free-recalled, was diminished in group A compared with C, with no differences with group NA. General cognition and incidental learning were related to declarative memory function exclusively in long-COVID groups. Long-COVID groups presented lower long-term retention of verbal declarative memory than controls in recall tests but no differences in recognition tests. No group differences were found in the acquisition of procedural memory. However, long-term retention of this memory was worse in group A as compared to the NA and C groups, respectively, when errors and time of execution were considered. Conclusion: Findings support that consolidation of both procedural and declarative memories is more affected than the acquisition of these memories in long-COVID patients, who are also more vulnerable to deficits in delayed recall than in recognition of declarative memories. Deficits in the consolidation of procedural memory and immediate recall of declarative information are especially relevant in long-COVID participants with anosmia. This indicates that anosmia in COVID-19 could be associated with a long-term dysfunction of the limbic system.

Memory alterations after COVID-19 infection: a systematic review.

SARS-CoV-2 infection has a wide range of both acute and long-term symptoms. Memory alterations have been frequently reported in studies that explore cognition. The main objective of the systematic review is to update and further analyze the existing evidence of objective memory impairments in long-COVID-19 considering sample and study design characteristics, as well as to explore associations between memory performance and their epidemiological, clinical, and pathological features. A total of 13 studies were identified by searching in PubMed, Web of Science, and PsycInfo databases up to May 6, 2022. Most studies evaluated verbal component of memory in the short-term and long-term recall up to 30 min and mainly performed a single assessment completed at 4-6 months after the infection. The samples mainly consisted of middle-aged adults that required hospitalization. Samples were not stratified by sex, age, and severity. Poor verbal learning was reported in most cases (6-58%), followed by deficits in long-term (4-58%) and short-term (4-37%) verbal memory. Visuospatial component of memory was studied less than verbal component, showing impairment of long-term retention of visual items (10-49%). COVID-19 severity in the acute stage was not systematically associated with poor memory performance. Verbal memory deficits were associated with anxiety and depression. The existing literature on objective memory assessment in long-COVID suggests further research is warranted to confirm memory dysfunction in association with epidemiological, pathological, and clinical factors, using both verbal and visuospatial tests, and exploring in deep long-term memory deficits.

Functional near-infrared spectroscopy in the neuropsychological assessment of spatial memory: A systematic review.

Functional near-infrared spectroscopy (fNIRS) is a non-invasive optical imaging technique that employs near-infrared light to measure cortical brain oxygenation. The use of fNIRS has increased exponentially in recent years. Spatial memory is defined as the ability to learn and use spatial information. This neuropsychological process is constantly used in our daily lives and can be measured by fNIRS but no research has reviewed whether this technique can be useful in the neuropsychological assessment of spatial memory. This study aimed to review empirical work on the use of fNIRS in the neuropsychological assessment of human spatial memory. We used four databases: PubMed, PsycINFO, Scopus and Web of Science, and a total of 18 articles were found to be eligible. Most of the articles assessed spatial or visuospatial working memory with a predominance in computer-based tasks, used fNIRS equipment of 16 channels and mainly measured the prefrontal cortex (PFC). The studies analysed found linear or quadratic relationships between working memory load and PFC activity, greater activation of PFC activity and worse behavioural results in healthy older people in comparison with healthy adults, and hyperactivation of PFC as a form of compensation in clinical samples. We conclude that fNIRS is compatible with the standard neuropsychological assessment of spatial memory, making it possible to complement behavioural results with data of cortical functional activity.

Evaluation of an Augmented Reality Application for Learning Neuroanatomy in Psychology.

Neuroanatomy is difficult for psychology students because of spatial visualization and the relationship among brain structures. Some technologies have been implemented to facilitate the learning of anatomy using three-dimensional (3D) visualization of anatomy contents. Augmented reality (AR) is a promising technology in this field. A mobile AR application to provide the visualization of morphological and functional information of the brain was developed. A sample of 67 students of neuropsychology completed tests for visuospatial ability, anatomical knowledge, learning goals, and experience with technologies. Subsequently, they performed a learning activity using one of the visualization methods considered: a 3D method using the AR application and a two-dimensional (2D) method using a textbook to color, followed by questions concerning their satisfaction and knowledge. After using the alternative method, the students expressed their preference. The two methods improved knowledge equally, but the 3D method obtained higher satisfaction scores and was more preferred by students. The 3D method was also more preferred by the students who used this method during the activity. After controlling for the method used in the activity, associations were found between the preference of the 3D method because of its usability and experience with technologies. These results found that the AR application was highly valued by students to learn and was as effective as the textbook for this purpose.

SLAM-based augmented reality for the assessment of short-term spatial memory. A comparative study of visual versus tactile stimuli.

The assessment of human spatial short-term memory has mainly been performed using visual stimuli and less frequently using auditory stimuli. This paper presents a framework for the development of SLAM-based Augmented Reality applications for the assessment of spatial memory. An AR mobile application was developed for this type of assessment involving visual and tactile stimuli by using our framework. The task to be carried out with the AR application is divided into two phases: 1) a learning phase, in which participants physically walk around a room and have to remember the location of simple geometrical shapes; and 2) an evaluation phase, in which the participants are asked to recall the location of the shapes. A study for comparing the performance outcomes using visual and tactile stimuli was carried out. Fifty-three participants performed the task using the two conditions (Tactile vs Visual), but with more than two months of difference (within-subject design). The number of shapes placed correctly was similar for both conditions. However, the group that used the tactile stimulus spent significantly more time completing the task and required significantly more attempts. The performance outcomes were independent of gender. Some significant correlations among variables related to the performance outcomes and other tests were found. The following significant correlations among variables related to the performance outcomes using visual stimuli and the participants' subjective variables were also found: 1) the greater the number of correctly placed shapes, the greater the perceived competence; 2) the more attempts required, the less the perceived competence. We also found that perceived enjoyment was higher when a higher sense of presence was induced. Our results suggest that tactile stimuli are valid stimuli to exploit for the assessment of the ability to memorize spatial-tactile associations, but that the ability to memorize spatial-visual associations is dominant. Our results also show that gender does not affect these types of memory tasks.

Wayfinding Strategy and Gender - Testing the Mediating Effects of Wayfinding Experience, Personality and Emotions.

BACKGROUND: Individual differences have been seen to play a key role in spatial orientation. Gender implications have been previously described but little is known about how other variables, such as wayfinding anxiety, emotional difficulties and wayfinding experience can mediate this relationship. METHODS: A group of 269 participants were involved in this study and completed questionnaires on their self-reported allocentric orientation strategy, wayfinding experience and satisfaction with the ability for wayfinding. Emotional outcomes were also investigated: spatial and trait anxiety, neuroticism, difficulties in emotion regulation, and personal safety. First, a principal component analysis was conducted and the studied variables were grouped into four components: outdoor wayfinding experience, wayfinding-related fear, emotional difficulties, and effective wayfinding skill. Afterwards, structural equation modelling was performed, using the MPLUS statistical program. RESULTS: The results showed that gender constitutes a predictor for using an effective wayfinding skill and for feeling wayfinding-related fear. However, outdoor wayfinding experience, wayfinding-related fear and emotional difficulties did not mediate the relationship between effective wayfinding skill and gender. CONCLUSION: These results highlight the differential contribution of gender in the emotions that are experienced during spatial orientation and emotions that are related to other types of situations. The limitations, strengths and theoretical implications of the proposed model are discussed. Further investigation is needed in order to understand the role of emotions in spatial orientation.

Memory for Object Location in Augmented Reality: The Role of Gender and the Relationship Among Spatial and Anxiety Outcomes.

The potential of augmented reality (AR) technology for the study of spatial memory and orientation is a new research field. AR defines systems that attempt to enhance the user's experience with the physical world. In our app, we enhance the sense of sight by adding interactive 3D elements to the real environment. Our app can be used in any real environment so that the experimental conditions during the tasks and the way in which an individual navigates are similar to those used in real life. With AR, the experimenter has a high level of control of the task and can store the participant's responses accurately. The classical factors that influence an individual's performance on virtual spatial tasks are gender and cognitive factors. The influence of emotional factors on spatial performance has been studied more recently. Since AR tasks for the study of spatial memory and spatial orientation are new developments, little is known about the factors that are related to performance on tasks of this type. In our study, we tested 46 young adults (26 women) in an AR object-location task that was performed in a building. The participants had to memorize the position of eight virtual objects while they were walking through the environment. We also assessed the participants' performance on an object-recall task, a map-pointing task, and a paper-and-pencil spatial orientation task. The self-reported importance of different spatial strategies for wayfinding and the levels of trait anxiety and wayfinding anxiety were also evaluated. Our findings indicate that men performed better on the spatial paper-and-pencil test and spent more time completing the learning phase of the AR task. The spatial memory for the location of the objects in AR and on the map correlated positively. Anxiety was related to individual differences in the self-reported use of a spatial orientation strategy, but the association among them was weak. Trait anxiety was positively related to the time employed by the participants during the learning phase of the AR task, whereas wayfinding anxiety correlated negatively with the preference for an orientation strategy. Our results highlight the importance of anxiety in spatial orientation.

Adult social isolation leads to anxiety and spatial memory impairment: Brain activity pattern of COx and c-Fos.

Social isolation during adulthood is a frequent problem that leads to a large variety of adverse emotional and cognitive effects. However, most of the social isolation rodent procedures begin the separation early post-weaning. This work explores locomotor activity, anxiety-like behaviour, and spatial working memory after twelve weeks of adult social isolation. In order to study the functional contribution of selected brain areas following a working memory task, we assessed neuronal metabolic activity through quantitative cytochrome oxidase histochemistry and c-Fos immunohistochemistry. Behaviourally, we found that isolated animals (IS) showed anxiety-like behaviour and worse working memory than controls, whereas motor functions were preserved. Moreover, IS rats showed lower levels of learning-related c-Fos immunoreactivity, compared to controls, in the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and nucleus accumbens shell. In addition, the IS group showed lower neuronal metabolic activity in the mPFC, VTA, and CA1 subfield of the hippocampus. These results indicate that twelve weeks of social isolation in adult rats leads to different behavioural and brain alterations, and they highlight the importance of social support, not only in development, but also in adulthood.

A Virtual Object-Location Task for Children: Gender and Videogame Experience Influence Navigation; Age Impacts Memory and Completion Time.

The use of virtual reality-based tasks for studying memory has increased considerably. Most of the studies that have looked at child population factors that influence performance on such tasks have been focused on cognitive variables. However, little attention has been paid to the impact of non-cognitive skills. In the present paper, we tested 52 typically-developing children aged 5-12 years in a virtual object-location task. The task assessed their spatial short-term memory for the location of three objects in a virtual city. The virtual task environment was presented using a 3D application consisting of a 120″ stereoscopic screen and a gamepad interface. Measures of learning and displacement indicators in the virtual environment, 3D perception, satisfaction, and usability were obtained. We assessed the children's videogame experience, their visuospatial span, their ability to build blocks, and emotional and behavioral outcomes. The results indicate that learning improved with age. Significant effects on the speed of navigation were found favoring boys and those more experienced with videogames. Visuospatial skills correlated mainly with ability to recall object positions, but the correlation was weak. Longer paths were related with higher scores of withdrawal behavior, attention problems, and a lower visuospatial span. Aggressiveness and experience with the device used for interaction were related with faster navigation. However, the correlations indicated only weak associations among these variables.

MnemoCity Task: Assessment of Childrens Spatial Memory Using Stereoscopy and Virtual Environments.

This paper presents the MnemoCity task, which is a 3D application that introduces the user into a totally 3D virtual environment to evaluate spatial short-term memory. A study has been carried out to validate the MnemoCity task for the assessment of spatial short-term memory in children, by comparing the children's performance in the developed task with current approaches. A total of 160 children participated in the study. The task incorporates two types of interaction: one based on standard interaction and another one based on natural interaction involving physical movement by the user. There were no statistically significant differences in the results of the task using the two types of interaction. Furthermore, statistically significant differences were not found in relation to gender. The correlations between scores were obtained using the MnemoCity task and a traditional procedure for assessing spatial short-term memory. Those results revealed that the type of interaction used did not affect the performance of children in the MnemoCity task.

Augmented reality for the assessment of children's spatial memory in real settings.

Short-term memory can be defined as the capacity for holding a small amount of information in mind in an active state for a short period of time. Although some instruments have been developed to study spatial short-term memory in real environments, there are no instruments that are specifically designed to assess visuospatial short-term memory in an attractive way to children. In this paper, we present the ARSM (Augmented Reality Spatial Memory) task, the first Augmented Reality task that involves a user's movement to assess spatial short-term memory in healthy children. The experimental procedure of the ARSM task was designed to assess the children's skill to retain visuospatial information. They were individually asked to remember the real place where augmented reality objects were located. The children (N = 76) were divided into two groups: preschool (5-6 year olds) and primary school (7-8 year olds). We found a significant improvement in ARSM task performance in the older group. The correlations between scores for the ARSM task and traditional procedures were significant. These traditional procedures were the Dot Matrix subtest for the assessment of visuospatial short-term memory of the computerized AWMA-2 battery and a parent's questionnaire about a child's everyday spatial memory. Hence, we suggest that the ARSM task has high verisimilitude with spatial short-term memory skills in real life. In addition, we evaluated the ARSM task's usability and perceived satisfaction. The study revealed that the younger children were more satisfied with the ARSM task. This novel instrument could be useful in detecting visuospatial short-term difficulties that affect specific developmental navigational disorders and/or school academic achievement.

Reduced cytochrome oxidase activity in the retrosplenial cortex after lesions to the anterior thalamic nuclei.

The anterior thalamic nuclei (ATN) make a critical contribution to hippocampal system functions. Growing experimental work shows that the effects of ATN lesions often resemble those of hippocampal lesions and both markedly reduce the expression of immediate-early gene markers in the retrosplenial cortex, which still appears normal by standard histological means. This study shows that moderate ATN damage was sufficient to produce severe spatial memory impairment as measured in a radial-arm maze. Furthermore, ATN rats exhibited reduced cytochrome oxidase activity in the most superficial cortical layers of the granular retrosplenial cortex, and, to a lesser extent, in the anterior cingulate cortex. By contrast, no change in cytochrome oxidase activity was observed in other limbic cortical regions or in the hippocampal formation. Altogether our results indicate that endogenous long-term brain metabolic capacity within the granular retrosplenial cortex is compromised by even limited ATN damage.

Visual working memory in deaf children with diverse communication modes: improvement by differential outcomes.

Although visual functions have been proposed to be enhanced in deaf individuals, empirical studies have not yet established clear evidence on this issue. The present study aimed to determine whether deaf children with diverse communication modes had superior visual memory and whether their performance was improved by the use of differential outcomes. Severely or profoundly deaf children who employed spoken Spanish, Spanish Sign Language (SSL), and both spoken Spanish and SSL modes of communication were tested in a delayed matching-to-sample task for visual working memory assessment. Hearing controls were used to compare performance. Participants were tested in two conditions, differential outcome and non-differential outcome conditions. Deaf groups with either oral or SSL modes of communication completed the task with less accuracy than bilingual and control hearing children. In addition, the performances of all groups improved through the use of differential outcomes.

Acetylcholinesterase activity in an experimental rat model of Type C hepatic encephalopathy.

Patients with liver malfunction often suffer from hepatic encephalopathy, a neurological complication which can affect attention and cognition. Diverse experimental models have been used to study brain alterations that may be responsible for hepatic encephalopathy symptoms. The aim of the study was to determine whether cognitive impairment found in cirrhosis could be due to disturbance of acetylcholinesterase activity. Acetylcholinesterase activity was assessed in the brains of Wistar rats with thioacetamide-induced cirrhosis. The cirrhotic group displayed up-regulation of acetylcholinesterase levels in the entorrhinal cortex, anterodorsal and anteroventral thalamus and accumbens, whereas down-regulation was found in the CA1, CA3 and dentate gyrus of the hippocampus. Our results indicate that the experimental model of hepatic encephalopathy by chronic administration of thioacetamide presents alterations of acetylcholinesterase activity in brain limbic system regions, which play a role in attention and memory.

Memory performance and scopolamine: hypoactivity of the thalamus revealed by cytochrome oxidase histochemistry.

Spatial memory learning is related to the functioning of a neuronal circuit composed of cortical, hippocampal and diencephalic brain regions. The Morris water maze (MWM) is frequently used to assess spatial memory in rats. In this study, the neuronal functional activity of some brain limbic system regions after a memory task in adult male Wistar rats injected with scopolamine (1.0mg/kg, i.p.) was assessed using cytochrome oxidase (COx) histochemistry. The rats were trained following a working memory schedule in the MWM. A trained group injected with saline and an untreated control group were examined to compare changes in COx activity in the dorsal hippocampus, anterior thalamus, mammillary nuclei, prefrontal cortex and ventral tegmental area. The scopolamine-treated group showed an impairment of spatial learning. Also, a decrease in COx activity was found in this group as compared to the saline group in the anteroventral and anteromedial thalamic nuclei. Overall, these findings suggest that memory deficits induced by scopolamine may be due to impairment of the cholinergic function in the anterior thalamic nuclei.

Reversal learning impairment and alterations in the prefrontal cortex and the hippocampus in a model of portosystemic hepatic encephalopathy.

Patients with liver dysfunction often suffer from hepatic encephalopathy (HE), a neurological complication that affects attention and memory. Various experimental animal models have been used to study HE, the most frequently used being the portocaval shunt (PCS). In order to determine brain substrates of cognitive impairment in this model, we assessed reversal learning and c-Fos expression in a rat model of portosystemic derivation. PCS and sham-operated rats (SHAM) were tested for reversal learning. Brains were processed for c-Fos immunocytochemistry. The total number of c-Fos positive nuclei was quantified in the prefrontal cortex and hippocampus. The spatial reference memory task showed no differences between groups in escape latencies. The no-platform probe test showed that both the PCS and the SHAM learned the location of platform. However, the PCS group perseverated in the old target during reversal. The PCS group presented less c-Fos- positive cells in prelimbic cortex, CA1 and dentate gyrus of the dorsal hippocampus than SHAM. Overall, these results suggest that this specific model of portosystemic hepatic encephalopathy produces reversal learning impairment that could be linked to dysfunction in neuronal activity in the prefrontal cortex and hippocampus.

Assessment of the global intelligence and selective cognitive capacities in preterm 8-year-old children.

The aim of this study was to assess various cognitive abilities such as attention, IQ, reasoning, and memory related to academic achievement in 8- and 9-year-old preterm children. A total of 141 children were assessed. The preterm group (=37 weeks) comprised 63 children and was compared to 78 full-term children. Attention was evaluated using the d2 Selective Attention test, and the IQ by the L-M form of the Stanford-Binet Intelligence Scale, establishing a profile of abilities: perception, memory, comprehension, reasoning, and verbal fluency. Significant differences in IQ were found between the preterm and full-term children. Of the cognitive abilities assessed, the only significant differences were found in verbal fluency, with preterm boys showing lower verbal fluency scores than full-term children. In conclusion, all preterm groups have attention ability similar to that of full-term children. However, preterm children obtain lower scores in intelligence measures. In addition, preterm boys have verbal fluency difficulties. Taking into account the increase in preterm births, suitable intervention programs must be planned to attend the difficulties found.