A Mini-Review on Conjugated Polymer-Based Fluorescence Probes for Bilirubin Detection

Introduction

Bilirubin is a bile pigment originating in two ways in the human body. The first one is hemoglobin disintegration (75%-85%), and the second one is myoglobin and cytochromes degradation (15- 25%).[1,2] Generally, normal level of bilirubin concentration in the blood range from 0.3 to 0.19 miligrams per 100 mL [3,4]. If the concentration of bilirubin exceeds the mentioned appropriate concentration, it is toxic for the human being; it may lead to jaundice and brain hemorrhage due to its accumulation on body organs. A low concentration of bilirubin is also harmful and causes coronary heart diseases [5-8]. For the detection of bilirubin, several techniques were developed by the researchers, including the Chemiluminescence based techniques [9], spectroscopic techniques, some diazo reactions, [10,11], and polarographic techniques [12]. Due to a lack of adequate selectivity and affectability, the researcher adopted different fluorescence techniques for bilirubin detection. Fluorescence Techniques are unique because of their simplicity, quick response, and economic nature. Various fluorescence probes like metal-organic framework (MoF) [13], quantum dots, and conjugated polymer (CPs) [14-17] have been reported to detect bilirubin. CPs have arisen as a flexible category of nanomaterials with incredible capability in biosensing and bioimaging. These CPs are soluble in the aqueous phase, appropriate for bio application. The solubility in water of rigid CPs is achieved due to functionalized with either nonionic neutral side chains or charged side chains. The polymer’s backbone exhibits a hydrophobic nature, compensated by the hydrophilic pendant groups, enhancing the solubility and interacting with the specifically targeted analytes. Due to the nonspecific interaction of nonbiological and multiple biological substances like DNA, RNA, and protein, selectivity is compromised. At the same time, conjugated polymer carried charged pendants, which can bind with the serum protein differentially. This report aims to provide information regarding conjugated polymer-based fluorescence Techniques for bilirubin detection.

Bilirubin Visual Sensing Via Self-Assembled Polyfluorenes [14]

This research report designed and synthesized two bulky appended containing polymers via Suzuki coupling polymerization, one homopolymer (PDP-PF) and the second copolymer (PDPPF-co- Ph), respectively. The sensing of both polymers toward bilirubin was shown by the FRET-based bilirubin green emission and polymer fluorescence quenching as presented in Figure 1. With the help of a UV lamp, it can be easily observed that the change in color from blue to green is due to bilirubin addition. The PDPPF-co-Ph polymer’s porous spherical assembly is able to adsorb analytes better than the simple micellar assembly, which produces an excellent spectral overlap and greater efficiency for FRET -energy transfer. An unbound bilirubin sensing was performed in Water/THF mixture to maintain the equilibrium between bound (water-soluble) and unbound (THF- soluble). The coupling between bilirubin emission increment and polyfluorene quenching emission creates this approach, appropriate and adaptable for the fluorimetric emission color change-based sensor. Fluorescence quenching efficiency of structural analogues of bilirubin like porphyrin and biliverdin showed poor; due to this reason, these new polymers can highlight the sensitivity and selectivity of the FRET-based sensing in the context of bilirubin.

Figure 1: FRET induced mechanism of bilirubin sensing in PDP-PF and PDPPF-co-Ph. Reproduced with permission from Ref. [14] Copyright (2013) American Chemical Society.

Bilirubin Sensing in Human Serum Via Fluorescent Probe Based on Polyfluorene: [15]

Senthilkumar et al.; designed a new conjugated polymer-based fluorescence sensing technique to detect bilirubin in water and human blood serum. This process acts on the FRET and plays a vital role in obtaining the emission of FRET mediated bilirubin which can change the blue color into green. This new polymer was synthesized in a two-step. In the first step, polyfluorenes are functionalized with glucuronic acid side chains; this is a click chemistry-based cross-coupling reaction followed by a palladium catalyst. The new polymer polyfluorenes attached with the appendage of D-glucuronic acid provide the selective sensing of bilirubin in the human blood serum without sticking with any serum protein because of its nonreceptor nature towards to protein as given in Figure 2. The tendency to form large aggregates in the serum by the homopolymer was supported by Dynamic light scattering (DLS). DLS is conducted in an aqueous medium as well as in human serum. In the aqueous medium, PF-GlcA showed a 330 nm particle size while PF-Ph-GlcA exhibited a small particle size of 110nm. With the help of the DLS result, it can be understood that a high volume of glucuronic acid with homopolymer PF-GlcA shows the tendency to stick to the protein, which is hydrophilic; this leads to the aggregation of polymer in the serum. So that in the design of the polymer, the glucuronic acid appendage should be optimum to avoid the proteins adherence. Interaction between the bilirubin and appendage of D-glucuronic acid of the polymer was identified with the help of an isothermal titration calorimeter. During the interaction, bilirubin act as a ligand, and polymer acts as a macromolecule; the result of this interaction produces heat which the ITC measured. At the end of the analysis, results reveal that the interaction between bilirubin and polymer is exothermic in nature. The stability of the polymer in human serum was found excellent under extreme basic conditions. Despite the various interferences like biliverdin, cholesterol, glucose, metal ions, and proteins in the human serum, the polymer exhibits high selectivity and sensitivity towards free bilirubin. This novel polymer could sense the bilirubin at the nano-level (∼150 nm).

Figure 2: FRET-induced Mechanisms of bilirubin sensing in human serum using polymer PF-Ph-GlcA. Reproduced with permission from Ref.xx Copyright (2015) American Chemical Society.

Bilirubin Detection Via “Turn-On” Fluorescence Using Water- Soluble Conjugated Polymer [16]

In this report, a conjugated polymer was designed and synthesized, which is water-soluble named PF-BT-GlcA poly(fluorenes-altbenzothiadiazole) used for turn sensing of bilirubin as indicated in Figure 3. The synthesis of the polymer was carried out in twostep, in the first step was performed via Suzuki cross-coupling polymerization reaction between glucuronic acid-functionalized fluorene monomer and benzothiadiazole bisboronic ester and produced the polymer PF-bT-GlcP. In the 2nd step, deprotection of PF-BT-GlcP takes place, and the final product PF-BT-GlcA was obtained as a polymer. The GPC data and NMR data indicate the deprotonation. After the preparation of the polymer, the bilirubin detection is performed in water. The polymer’s solubility in an aqueous medium plays a crucial role, essential for sensing studies. As per the emission and absorption spectra of polymer, with increasement in bilirubin concentration, increasement in the absorption and emission was observed at 520nm and 462nm, respectively. After successful detection in water, further studies were done with the human serum. During this experiment, many obstructions were included in the human serum, such as cholesterol, hemoglobin, protein, triglyceride, and metal ions. The bilirubin detection in human serum was led by the polymer PFBT- GlcA blended in human serum with free bilirubin; the emission spectra illustrate a similarly sharp peak at 520nm. The report concludes the work by demonstrating the turn-on fluorescence sensing of bilirubin using conjugated polymer.

Figure 3: FRET-induced mechanism of bilirubin sensing using polymer PF-BT-GlcA in human serum. Reproduced with permission from Ref. [16] Copyright (2022) American Chemical Society.

Bilirubin Detection in Human Serum Via Fluorescence Probe Based on Conjugated Polymer Nanoparticle [17]

In this research work Iyer et.al., designed and synthesized a polyfluorene derivative conjugated polymer poly1,1′-((2,7- dimethyl-9H fluorene-9,9-diyl) bis(hexane-6,1-diyl) bis(1Hbenzo[ d]imidazole) (PFBZ) with special receptor via oxidative coupling polymerization reaction. Further, this polymer utilizes for the detection of bilirubin. The complexation between bilirubin and PFBZ polymer and FRET is initially responsible for the sensing mechanism. PFBZ polymer can be utilized in human blood serum for the bilirubin; results were appropriate with a very low standard deviation. In the aqueous medium, PFBZ polymer forms nanoparticles spontaneously and can exhibit the limit of detection of 6.9 pM. After obtaining good results, the researcher developed a fabricated paper-based fluorescence test kit that seems very simple and gives quick response times as shown in Figure 4.

Figure 4: Fluorescence kit based on the color of paper under a UV lamp (365nm) after adding different concentrations of bilirubin. Reproduced with permission from Ref [17] Copyright (2022) American Chemical Society.

Conclusion

Different researchers successfully designed and synthesized the conjugated polymers via click chemistry and Suzuki polymerization. The further analysis provides information regarding the particle size and interaction between bilirubin and polymer. The analysis results reveal bilirubin acts as a ligand and polymer acts as macromolecules. The FRET emission and polymer fluorescence quenching demonstrate the sensing efficiency of the polymer towards the bilirubin. Table 1 summarizes the various conjugated polymer based fluorescent probe for the detection of bilirubin with different LOD values. Conjugated polymers are excellent materials for the detection of bilirubin in water as well as human serum as low as 6.9 pM.

Table 1: Comparison of some previously reported conjugated polymer-based fluorometric sensors for bilirubin detection.

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Evaluation of Freeze-Drying Process of Aqueous Bovine Serum Albumin Dispersions

Introduction

Freeze drying [FD], also known as lyophilization, is a technical process widely used for pharmaceutical preparations for many reasons, such as for improving stability and long-term storage stability, especially for temperature sensitive drugs, such as proteins, hormones, and vaccines. It is used now as a novel method for loading drugs and targeting sick cells [1-3]. Suitable parameters of process application allow us to obtain best quality products compared to products dried with traditional methods. Very good physical and chemical properties make this method the best for drying exclusive products [2,4]. Freeze drying is the process that could take days to finish, and that one of its principal disadvantages. The time depends mainly on the design and optimization of the FD process. Freeze drying works on the principle of sublimation, where free water present in the material under process is frozen and directly converted into vapor state without entering the liquid phase [4,5]. A typical freeze-drying process consists of three stages which is: Freezing, primary drying, and secondary drying [2,6,7]. The freezing stage typically takes several hours to finish. Annealing is after freezing is simply holding the product at a temperature above the final freezing temperature for a defined period to crystallize the potentially crystalline components in the formulation during the freezing stage [1,2,8]. An annealing step is frequently necessary to allow efficient crystallization of the crystalline bulking agent, such as mannitol or glycine [1,2]. Primary drying, or ice sublimation, begins whenever the chamber pressure is reduced, and the temperature is raised to supply the heat removed by ice sublimation. The primary drying stage is the longest stage of freeze drying and optimization of this stage has a large impact on process economics [4,5]. Secondary drying is the stage where water is desorbed from the freeze concentrate at elevated temperature and low pressure. Secondary drying normally takes only hours, and the opportunity for time reduction by process optimization is limited [6-8].

The stability of the drug during freeze drying and storage, and the duration of the cycle are the two major considerations for freeze drying process optimization, although other parameters such as excipients, pressure, and primary and secondary drying temperatures have a great role on the FD process design [9- 11]. To develop a successful protein formulation using freezedrying procedure, physical properties, such as glass transition temperature (Tg) and residual moisture content, and operational parameters, should be considered, as well as the selection of suitable excipients [9,10]. Sugars, for example, as sucrose, lactose, mannitol and trehalose proved to stabilize various proteins during FD. They are the cryoprotectants of choice, and their stabilization mechanisms are well described by the water replacement and vitrification/ particle isolation theories [7,9,11]. Another example, PEG conjugation masks the protein’s surface during FD and increases the molecular size of the polypeptide, thus preventing the approach of antibodies or antigen processing cells and reducing the protein’s degradation [12]. The benefits of freeze-drying mainly for conserving proteins derived from its reduction in the deleterious chemical reactions as the diffusion of protein molecules is greatly inhibited in the vitrified solid-state [11,12]. Freeze-drying technology is not only used to prepare stable proteins, but also it is suitable for a wide range of applications, such as manufacturing biocarriers (e. g. Artificial oxygen carriers to reduce the need of patients for erythrocyte concentrates) [13], or for preserving the characteristics of the initial pharmaceuticals (e. g. Attenuated virus vaccines) [14], or for conserving natural biomaterials and cells in a dry form [15,16], or conserving food with best quality [17], or for increasing drug solubility [6,8,18] or for drug targeting and controlling release [19-21]. Bovine Serum Albumin (BSA), was chosen in our study as a model of protein. It is a non-glycosylated protein of 66 kDa, produced by the liver, and it is the most abundant protein in plasma. When it is heated to 50°C or above, albumin quite rapidly forms hydrophobic aggregates which do not revert to monomers upon cooling [4,5]. The aim of this work was to prepare stable freeze-dried powder of albumin, and to evaluate the optimal freeze-drying formulation.

Materials and Methods

Bovine Serum Albumin (BSA, fraction V) was purchased from Biowest- USA. Glutaraldehyde 8% aqueous solution, mannitol, sucrose, and lactose were purchased from Sigma- Germany. Bradford reagent, ethanol, phosphate buffered saline, sodium chloride, were purchased from Merck- Germany. All other chemicals and reagents were of analytical grade and used as received. Water was used as ultra-pure.

Preparation of BSA Suspension

200 mg BSA in 10 ml of 10 mM NaCl solution, adjusted to pH= 7.4 with phosphate buffer, then were transformed into nanoparticles by continuous addition of 8 ml ethanol 96% under constant stirring at room temperature. After obtaining a clear solution, 235 ml of glutaraldehyde solution was added with stirring for 24 hours at room temperature, then the resulting particles were centrifuged (using Sigma 3-16 KL centrifuge- Germany), then separated and redispersed to 10 ml of water to obtain a concentration of 2% of BSA only before lyophilization process [4].

Preparation of Sugar Solutions

Specific quantities of mannitol, sucrose and lactose were separately dissolved in water to obtain concentrations of 2,4, and 6%.

Preparation of Albumin-Sugar Suspensions

10 ml of freshly prepared sugar solution was added, before FD, to 10 ml of freshly prepared BSA suspension with continuous stirring for 2 h.

Freeze- Drying Process

1% and 2% of BSA suspension, sugar solutions of 1%, 2%, 3%, and mixtures of them were freeze dried as the following conditions:

a) Freezing: at -60 ͦC: 3 hours.

b) Annealing: at -40 ͦC: 6 hours.

c) Primary drying: -60 ͦC →-30 ͦC. Pressure: 0.1 mbar.

d) Secondary drying: -30 ͦC →+25 ͦC. Pressure: 0.1 mbar.

e) Using FreeZone Plus 2.5 Liter Cascade Freeze Dry Systems

of Labconco apparatus. (Labconco Corp. Kansas City. MI. USA).

Determination of the Residual Moisture

The lyophilized samples (about 100 mg) were analyzed by a Karl Fischer titrator (Mettler DL 18- Mettler Toledo- USA).

Determination of Size and Size Distribution

The samples before lyophilization, were reconstituted by 10 ml of water, and analyzed by Coulter LS230-France, and this also applied to the lyophilized samples after preparation, and during storage.

Determination of yield%

Yield was determined by microgravimetry. (50 μl) of particles was put in a pan and dried for 2 h at 80 ͦC. The pans were weighed and the difference between the empty and filled pans was calculated [4,8].

BSA- Assay (Bradford Assay Method)

The Bradford protein assay, named after its developer Marion M. Bradford, is specifically used to calculate the concentration of total protein in a sample or solution.

Bradford Reagent Preparation: 100 mg of the reagent was dissolved in 50 ml of 95% ethanol and 100 ml of 85% phosphoric acid, mixed until completely dissolved, then water was added to 850 ml in volume.

BSA Standards Preparation: A set of 7 concentrations of BSA: 2, 1.5, 1, 0.75, 0.5, 0.25, 0.125 mg/ml) each was prepared by dissolving the accurate weight of BSA in the phosphate buffer (pH= 7.4), then 1.5 ml of Bradford reagent was added and incubated 10 min. The absorbance was measured at 595 nm (using T80 spectrophotometer from PG Instruments- UK), and the standard curve was performed. The blank, which is used to set the instrument to 0 absorbance was prepared in the same buffer as in the tested samples without BSA [22].

Sample Preparation: 20 mg of lyophilized BSA was dissolved in 3 ml of phosphate buffer (pH= 7.4) with stirring for 10 minutes at room temperature, then 1.5 ml of Bradford reagent was added, and the absorbance was measured [22].

Long- Term Stability

The lyophilized powders were stored at 2-8 ͦC, and 25 ͦC /60 RH for three months. Samples were characterized every month with regard of particle size, size distribution, and BSA assay. Table 1 below, shows the twelve studied formulations.

Statistical Analysis

All the data were presented as mean ± standard deviation (SD) of three separate experiments, and analyzed with t-test, p < 0.05 was indicative of significant difference between the test groups. The null hypothesis used in the statistical analyses propose there were no differences between certain characteristics of the generated data. When BSA was lyophilized without any excipient (in F1 and F2), the resulted powders were wet, sticky, and the residual moisture was nearly 5%. After reconstitution of the freeze-dried powder, large aggregates were produced (as shown in Table 2), and the cake appearance was visually evaluated especially in 2% BSA concentration. These observations ascertained the need of stabilizing the protein [4-6].

Results and Discussion

Table 2 shows the measurements of particles size: Z1 and Z2, before and after FD respectively, yield, and residual moisture. The obtained results indicated to the increasing in size of particles and decreasing in the residual moisture after freeze drying to almost the formulations. Aggregates were reduced as the particles size measurements showed, and that due to the stabilizing effect of the lactose sucrose, and mannitol. The formulations F7, F10, and F12 were selected after a statistical study for further investigations (long-term stability). These three formulations were stored either at a temperature = 4 ͦC or 25 ͦC / RH 60% for three months. The results of particles size and residual moisture are shown in Tables 3 & 4. As shown in Table 3, there is no considerable changes in size or residual moisture, which indicates the good storage conditions. The results showed, no considerable changes in size or residual moisture for F7 (Sucrose 2%), which indicates the good role of sucrose as a stabilizing agent in lyophilization process. The increase in particles size in F10 and F12 could be either because of mannitol crystallization or because of albumin aggregation. For further investigations, BSA assay in the three formulations F7, F10, and F12 was performed. BSA assay results for F7 (Figure 1) shows nearly the same results after three months of storage at 4 ͦC (97.5%), whereas a little bit less values at 25 ͦC (96.7%). This indicates no considerable change in albumin structure in both conditions, however the best values were when the temperature was at 4 ͦC. BSA assay results for F10 (Figure 2) shows nearly the same results after three months of storage at 4 ͦC (96.2%), but a decrease in values after three months of storage at 25 ͦC (from 96.5% to 92.1%). This indicates some changes in albumin structure or aggregations. BSA assay results for F12 (Figure 3) shows nearly the same results after three months of storage at 4 ͦC (96.7%), but a decrease in values after three months of storage at 25 ͦC (from 97.4% to 93.2%). This could be because of albumin aggregation or degradation.

Table 1: Freeze- dried BSA formulations.

Table 2: Particle size, yield, and residual moisture measurements.

Table 3: Particle size, and residual moisture measurements (Storage at 4 ͦC).

Table 4: Particle size and residual moisture measurements (Storage at 25 ͦC).

Figure 1: BSA assay for F7 during storage (T= 4 oC or 25 oC).

Figure 2: BSA assay for F10 during storage (T= 4 OC or 25 OC).

Figure 3: BSA assay for F12 during storage (T= 4 oC or 25 oC).

Conclusion

Freeze drying used to obtain stability of heat sensitive active pharmaceuticals during distribution and storage conditions. The present work shows that when BSA was lyophilized alone, without any excipient, large aggregates in the micrometer size range were observed. Stabilizing excipients in formulation, such as lactose, sucrose, and mannitol were necessary to obtain a stable freeze dried BSA both during the freeze-drying process, as well as for long term storage. Stability was assessed by aggregation state (particle diameter), residual moisture, and protein assay. The best results were with sucrose 2%, and storage temperature at 4 ͦC. In conclusion, successful lyophilization process must take into account two important factors, including the optimal formulation components and the understanding of critical parameters of the process.

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The Impact of Breastfeeding to Cognitive Functions in 5 Years Old Children

Introduction

Recently, we studied the impact of air pollution on cognitive development in 5 years old children during their prenatal period. The study was located to the Moravian-Silesian Region as it is considered the most polluted region in the Czech Republic by PM2.5 (particulate matter < 2.5 mm) and polycyclic aromatic hydrocarbons (PAHs) as B[a]P (benzo[a]pyrene] and control region Southern Bohemia. Two districts were selected: the more exposed district of Karvina (Northern Moravia) and the control district of Ceske Budejovice (Southern Bohemia) [1,2]. In the two cohorts of children, we did not observe any significant effect of prenatal PAH exposure on psychological cognitive tests results in the five years old children tested Blazkova, et al. [3], however, we observed the impact of oxidative damage associated with PM2.5 exposure during the prenatal period Blazkova, et al. [4]. Similar prospective cohort was followed in Krakow, Poland [5] for their cognitive development. Children were assessed at the age of 5 years by the Raven test (the Raven Colored Progressive Matrices, the RCPM test). According to the study results, prenatal exposure to PAHs higher than 17.96 ng/m3 decreased the RCPM scores [6]. Transplacental exposures to PAHs were related to a shorter head circumference, a lower birth weight, and a lower birth length which may be later related to lower cognitive functions and poorer school performance. In this cohort, Jedrychowski, et al. [7] observed the association between the cognitive achievements and breast feeding. Therefore, we also analyzed a possible relationship between the length of breastfeeding and cognitive development in the two cohorts of children mentioned, differing by a distinct level of air pollution: Karvina.PM2.5 mg/m3 37.7±14.7, B[a]P ng/m3 6.1±4.53, Ceske Budejovice PM2.5 mg/m3 17.1±4.8, B[a]P ng/ m3 1.19±1.28 [3].

Materials and Methods

Subjects

The cohorts were created in the summer 2013 and winter 2014 from newborns born in the Ceske Budejovice Hospital, Department of Obstetrics and Gynaecology and Department of Neonatology, and in the Karvina Hospital, Department of Obstetrics and Gynaecology and Department of Neonatology. Newborns were selected from the normal deliveries (38th-41st week) of non-smoking mothers, who signed a written consent. Cohorts included 99 newborns (summer) and 100 newborns (winter) in Ceske Budejovice, and 71 newborns (summer) and 74 newborns (winter) in Karvina. The study was approved by the Ethics Committee of both hospitals and the Institute of Experimental Medicine CAS in Prague. Between November 2018 and November 2019, 199 mothers from Ceske Budejovice district and 143 from Karvina district who provided samples from their children in 2013 and 2014, were approached to take part in psychological testing. Undertaking psychological test was optional. Out of the total amount of 342 potential subjects, 140 refused to take part in the study, and 31 were impossible to contact. In the present study, data from 80 children from Ceske Budejovice and 67 children from Karvina were collected. The final sample therefore included 147 children. This study was approved by the Ethical Committee of the Faculty of Health and Social Science, University of South Bohemia, Ceske Budejovice from June 30, 2017.

Measures of Child Visual-Motor Functioning and Intellect

To be able to examine the potential effect of breastfeeding on cognitive development in 5 years old children, two psychological assessment instruments were used, namely the Bender Visual Motor Gestalt test (the BG test) and the Raven Colored Progressive Matrices test (the RCPM test). From a variety of possible standardized tools, these two methods have been chosen considering the age of the tested children and the fact that measurement should have been successfully done in one session. Both methods are usually well received by children and help them adapt to the test situation. 5 years old children from our cohort were tested individually. The child psychologist who administered the psychological assessment instrument was unaware either of the fact whether the given child had been breastfed or how long did the breastfeeding took if relevant. In order to assess level of visual-motor functioning in 5 years old children, the Bender Visual Motor Gestalt test (BG test) was used. The test focuses on assessing motor functioning, visual perception, and potential developmental or neurological impairments in children and adults [8].

146 children at the age of 5 years completed the test. Each of our 5 years old children was presented with nine cards depicting different geometric shapes. The cards were presented individually and the tested children were asked to copy the design, trying to make the best reproduction possible. Test results were scored based on the organization and accuracy of the reproduction. This drawing test was well received by children and helped them considerably to get used to and feel comfortable with the test situation. Once the BG test was completed, the children were presented with a non-verbal intelligence test called Raven Colored Progressive Matrices (RCPM test) [9] that was also used in a similar study in Krakow, Poland [6]. The test has been developed and widely used for assessing reasoning and problem-solving ability in children between 5 and 11 years, including those suffering some kind of physical or mental impairment. The RCPM test consists of three sets of twelve matrix designs with increasing level of difficulty. 145 children at the age of 5 years completed the RCPM test.

Questionnaire for Mothers

Mothers engaged in the study provided us with information regarding social environment of the family, breastfeeding and eating habits, and child´s medical history. Similarly, the data regarding gestational age, birth weight, birth length, head circumference, and Apgar score were collected in order to be taken into account while analyzing psychological test results. Breast feeding details from maternal questionnaire has been in form of 3 particular questions: if the mother breastfed, how long she breastfed the baby fully and for how long the mother breastfed the child at least partially.

Statistical Analysis

The length of breastfeeding has been standardized into months and split into two groups, breast feeding up to 6 months and more than 6 months. Both groups have been compared with each other using Mann Whitney U-test in total and in particular groups by regions and children gender. Other potential values from the questionnaire have been tested as potential confounders – see in Table 1.

Results

The tested confounders are shown in Table 1. The district of Karvina differed from the district Ceske Budejovice as follows: mothers from Karvina were younger (p < 0.05), children length was shorter (p~0.001), maternal lower secondary education is more often (p < 0.05), higher education is less often (p < 0.01). The birth length and head perimeter was lower in girls vs. boys (p < 0.01) in both cohorts. No differences in the length and weight at 5 years was observed between Karvina and Ceske Budejovice as well as between boys and girls. The RCPM scores as well as the BG scores did not correlate either with respiratory or with all diseases in examined children during analyzed 5 years. The results of psychological tests affected by breastfeeding are presented in Table 2. For the RCPM test of full breastfeeding for 3 months, the correlation was positive in the RCPM test results in Karvina (p < 0.05) and in boys (p < 0.05). 6 months of full breast feeding correlated positively with higher results in the RCPM test (p < 0.05) and in Karvina (p < 0.05). Using the BG test, the full breastfeeding for 3 months correlated positively with higher test results in all study (p < 0.05), and in boys. 6 months of full breastfeeding correlated positively with better results in the BG test in all study (p < 0.001), Ceske Budejovice (p < 0.05), Karvina (p < 0.001), boys (p < 0.01) as well as girls (p < 0.05). Obtained results indicate generally more significant effect of full breastfeeding lasting 6 months and longer. It may be also speculated that the full breastfeeding may have more significant effect on future cognitive development in children living in district of Karvina which is more exposed to air-pollution Table 3. illustrates impact of maternal education levels on result of cognitive testing. Children of university graduated mothers have significantly higher level of RCPM score in all groups. For BG test score is this impact significant only for boys, but there is significantly lover score levels for mother’s lover secondary in all.

Table 1: Overview of tested confounders.

Note: Results of Mann Whitney U-test compare by region *) p ~ 0.05, **) p ~ 0.01, ***) p ~ 0.001 and by gender +) p ~ 0.05, ++) p ~ 0.01, +++) p ~ 0.001.

Table 2: Impact of breastfeeding on cognitive functions.

Note: Results of Mann Whitney U-test compared by Breastfeeding length *) p ~ 0.05, **) p ~ 0.01, ***) p ~ 0.001

Table 3: Impact of maternal education to cognitive functions.

Results of Mann Whitney U-test compared by Education level *) p ~ 0.05, **) p ~ 0.01, ***) p ~ 0.001

Discussion

Horta, et al. [10] reviewed the relationship between breastfeeding and intelligence. This meta/analyses shows that breastfeeding is related to improved performance in intelligence test. This conclusion is also supported by our results using the BG test. Jedrychowski, et al. [7] assessed the effect of exclusive breastfeeding for birth cohort in Krakow, which was exposed to higher concentrations of PAHs in polluted air. Their study provides evidence about the beneficial effect of exclusive breast feeding for 3 months on cognitive development of children at a preschool age, the benefit for children breastfed for > 6 months was more pronounced. Therefore, they support the WHO expert recommendation on exclusive breastfeeding for six months [11]. On the other hand, another Polish mother cohort study did not show that exclusive breastfeeding for > 6 month was associated with a psychomotor development in 1 year old children [12]. Von Stumm and Plomin [13] analyzed the benefits of breastfeeding on cognitive development in British twin study through adolescence up to 16 years. They proposed that breastfeeding was slightly associated with early life intelligence but not with later cognitive growth. Also Girard, et al. [14] did not observe the effect of breastfeeding > 6 moths on cognitive development of children at age 5 years. They suggested that earlier observed benefit for breastfeeding may not be maintained once children enter the school. Pang et al. study [15] advocated that nutrients in breast milk may improve general child cognition compared to infants fed formula only. Kim and Choi [16] studied associations between the breastfeeding and cognitive function in children up to 8 years. Children breastfed > 3 months scored significantly higher on communication and problem solving. Similarly, Lenehan, et al. [17] demonstrated in children breastfed > 2 months increased IQ and nonverbal IQ at 5 years of age.

Study by Lopez, et al. [18] identified a strong association between breastfeeding duration and general Ability scores in children aged 9 / 10 years, with the greatest effect found in those breastfed for more than 12 months. Longitudinal cohort study in Australia [19] examined the relationship between duration of breastfeeding and motor development outcomes at 10, 14, and 17 years. Breastfeeding for more than 6 months was positively associated with improved motor development outcomes. Our results suggest a positive effect of breastfeeding lasting 6 months and/or longer on cognitive development in 5 years old children. This effect was more pronounced in the BG test results focusing on visual-motor skills compared to the RCPM test results measuring problem-solving skills.

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Abducens Nerve and Facial Nerve Palsy in the Setting of an Extracranial Mass and Vascular Disease

Case Report

I present a case of a 59-year-old female patient who presented to a primary care clinic in August 2021 after she woke up from a nap with sudden onset horizontal binocular diplopia that worsened on far vision. She also reported a severe left side headache that worsened with loud noises. Gait instability was also noted. Physical exam revealed significant right eye esotropia and left eye CN 6 palsy which did not cross the midline on lateral gaze. Signs of right cranial nerve 7 palsy were seen with right side facial drop and ptosis apparent on inspection. Cranial nerves two and three were deemed to be intact as both pupils reacted equally to light and accommodation. She denied any history of trauma. At this initial visit, the etiology was suspected to be a viral auto-immune sequala and a steroid dose pack was prescribed. Her medical history is significant for hypertension, dyslipidemia, diabetes mellitus, and hypothyroidism. She has a 60-pack year smoking history. CTA in August 2021 was negative for any large vessel occlusion or hemorrhage. MRI was negative for any significant occlusion but revealed an extra-cranial mass, which measured 1cm and impacting the right trigeminal nerve. Subsequent follow up visits in August 2021 revealed no improve after corticosteroid therapy with persistent 6 and 7th nerve palsy, worsening alternating esotropia and further balance issues. No surgical intervention was made and a repeat MRI in 6 months was planned. At that time, the focus of treatment was to improve glycemic control, more tightly control lipids levels, and advise smoking cessation. Nearly three months to the date of onset, the patient awoke with restored lateral gaze and absent diplopia. Her facial palsy improved in subsequent weeks.

Discussion

Even in the presence of a facial palsy mass, vascular risk factors for patients with abducens nerve palsy must be evaluated. In adults, vascular disease constitutes a majority of abducens nerve palsy as shown by a retrospective chart review in 2014 [4]. Major risk factors for cranial atherosclerosis include diabetes mellitus, hypertension, metabolic syndrome, smoking, and a sedentary lifestyle [5]. The right extra-cranial mass was considered a possible etiology of both cranial neuropathies but was subsequently ruled out based on imaging and patient presentation. The patient’s right cranial nerve 7 palsy improved making the compression from a growing mass an unrealistic etiology. In relation to the left abducens nerve palsy, its contralateral location and sudden improvement highlighted the vascular nature of her etiology. A clearly defined etiology for facial palsies proves difficult to find. 70% of unilateral facial palsies are idiopathic with trauma, infection, and neoplasia as the remaining most common causes. In idiopathic cases, a viral prodrome period occurs before the onset of palsy [6].

The patient denied any preceding symptoms making a viral cause less likely but not impossible cause. The facial nerve has a robust vascular supply due to its thick epineurium; this vascular dependency proposes an ischemic connection to facial nerve palsy [7]. Although the facial nerve vascular supply has many anastomoses, this system can be compromised by diabetes mellitus, which is present in this case, especially at certain points along its tract that do have as proficient collateral perfusion as in the stylomastoid and petrosal branches [8]. Concurrent abducens and facial nerve palsies are a very uncommon finding. A 51-patient prospective study on bell’s palsy found that the most common concurrent cranial nerve palsies involved the trigeminal, glossopharyngeal, and hypoglossal nerves [9]. At this time, it is difficult to predict the long-term outcome for the patient presented in this case report but the prognosis for isolated abducens nerve palsy is encouraging. A 213-patient review found that 78.5% experience spontaneous recovery of their symptoms, with 36.6% by 8 weeks [10].

Conclusion

Patients that present with cranial neuropathies require a comprehensive vascular assessment in addition to the evaluation of neoplasms, trauma, and infectious causes. In the setting of a facial palsy, the etiology should not be immediately assumed to be of viral or auto-immune origin especially in diabetes mellitus, which predisposes the facial nerve to microvascular infarct. Multiple concurrent cranial neuropathies further stress the need for the evaluation of vascular risk factors.

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